US8697699B2 - Imidazopyrazine SYK inhibitors - Google Patents

Imidazopyrazine SYK inhibitors Download PDF

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US8697699B2
US8697699B2 US13/343,624 US201213343624A US8697699B2 US 8697699 B2 US8697699 B2 US 8697699B2 US 201213343624 A US201213343624 A US 201213343624A US 8697699 B2 US8697699 B2 US 8697699B2
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alkyl
imidazo
pyrazin
amino
optionally substituted
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Scott A. Mitchell
Peter A. Blomgren
Joseph Raker
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Kronos Bio Inc
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Gilead Connecticut Inc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
    • C07D487/04—Ortho-condensed systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
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    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
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    • A61P37/00—Drugs for immunological or allergic disorders
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    • A—HUMAN NECESSITIES
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • Spleen Tyrosine Kinase is a member of the Syk family of tyrosine kinases, and is a regulator of early B-cell development as well as mature B-cell activation, signaling, and survival.
  • Syk is a non-receptor tyrosine kinase that plays critical roles in immunoreceptor- and integrin-mediated signaling in a variety of cell types, including B cells, macrophages, monocytes, mast cells, eosinophils, basophils, neutrophils, dendritic cells, T cells, natural killer cells, platelets, and osteoclasts.
  • Immunoreceptors as described here include classical immunoreceptors and immunoreceptor-like molecules.
  • Classical immunoreceptors include B-cell and T-cell antigen receptors as well as various immunoglobulin receptors (Fc receptors).
  • Immunoreceptor-like molecules are either structurally related to immunoreceptors or participate in similar signal transduction pathways and are primarily involved in non-adaptive immune functions, including neutrophil activation, natural killer cell recognition, and osteoclast activity. Integrins are cell surface receptors that play key roles in the control of leukocyte adhesion and activation in both innate and adaptive immunity.
  • Syk is essential for B-cell activation through B-cell receptor (BCR) signaling.
  • BCR B-cell receptor
  • SYK becomes activated upon binding to phosphoryated BCR and thus initiates the early signaling events following BCR activation.
  • B-cell signaling through BCR can lead to a wide range of biological outputs, which in turn depend on the developmental stage of the B-cell.
  • the magnitude and duration of BCR signals must be precisely regulated.
  • Aberrant BCR-mediated signaling can cause disregulated B-cell activation and/or the formation of pathogenic auto-antibodies leading to multiple autoimmune and/or inflammatory diseases.
  • Mice lacking Syk show impaired maturation of B-cells, diminished immunoglobulin production, compromised T-cell-independent immune responses and marked attenuation of the sustained calcium sign upon BCR stimulation.
  • Protein-based therapeutics such as Rituxan developed to deplete B-cells represent an approach to the treatment of a number of autoimmune and inflammatory diseases.
  • Auto-antibodies and their resulting immune complexes are known to play pathogenic roles in autoimmune disease and/or inflammatory disease.
  • the pathogenic response to these antibodies is dependent on signaling through Fc Receptors, which is, in turn, dependent upon Syk. Because of Syk's role in B-cell activation, as well as FcR dependent signaling, inhibitors of Syk can be useful as inhibitors of B-cell mediated pathogenic activity, including autoantibody production. Therefore, inhibition of Syk enzymatic activity in cells is proposed as a treatment for autoimmune disease through its effects on autoantibody production.
  • Syk also plays a key role in Fc ⁇ RI mediated mast cell and eosinophil activation.
  • Syk is implicated in allergic disorders including asthma.
  • Syk binds to ITAM motifs on the phosphorylated gamma chain of Fc ⁇ RI via its SH2 domains and is essential for downstream signaling.
  • Syk deficiency in mice is associated with impaired IgE-mediated mast cell activation as marked by defective degranulation, leukotriene release, and cytokine secretion (e.g. TNF-alpha).
  • Pharmacologic agents that inhibit Syk activity have also been shown to inhibit mast cell degranulation, leukotriene release, and cytokine secretion in cell based assays.
  • Syk inhibitors have been shown to inhibit antigen-induced passive cutaneous anaphylaxsis, bronchoconstriction and bronchial edema in rats.
  • Treatment with Syk antisense oligonucleotides inhibits antigen-induced infiltration of eosinophils and neutrophils in an animal model of asthma.
  • Syk deficient eosinophils also show impaired activation in response to Fc ⁇ RI stimulation. Therefore, small molecule inhibitors of Syk will be useful for treatment of allergy-induced inflammatory diseases including asthma.
  • the inhibition of Syk activity can be useful for the treatment of allergic disorders, autoimmune diseases and inflammatory diseases such as: SLE, rheumatoid arthritis, multiple vasculitides, idiopathic thrombocytopenic purpura (ITP), myasthenia gravis, allergic rhinitis, chronic obstructive pulmonary disease (COPD), adult respiratory distress syndrome (ARDs) and asthma.
  • SLE rheumatoid arthritis
  • multiple vasculitides idiopathic thrombocytopenic purpura (ITP), myasthenia gravis, allergic rhinitis, chronic obstructive pulmonary disease (COPD), adult respiratory distress syndrome (ARDs) and asthma.
  • ITP idiopathic thrombocytopenic purpura
  • COPD chronic obstructive pulmonary disease
  • ARDs adult respiratory distress syndrome
  • inhibition of Syk activity may be useful in treating certain types of cancer, including B-cell lymphoma and leukemia.
  • compositions comprising at least one chemical entity described herein, together with at least one pharmaceutically acceptable vehicle chosen from carriers, adjuvants, and excipients.
  • Also provided is a method for treating a patient having a disease chosen from cancer, autoimmune diseases, inflammatory diseases, acute inflammatory reactions, and allergic disorders comprising administering to the patient an effective amount of at least one chemical entity described herein.
  • Also provided is a method for increasing sensitivity of cancer cells to chemotherapy comprising administering to a patient undergoing chemotherapy with a chemotherapeutic agent an amount of at least one chemical entity described herein, sufficient to increase the sensitivity of cancer cells to the chemotherapeutic agent.
  • Also provided is a method for inhibiting ATP hydrolysis comprising contacting cells expressing Syk with at least one chemical entity described herein in an amount sufficient to detectably decrease the level of ATP hydrolysis in vitro.
  • Also provided is a method for determining the presence of Syk in a sample comprising contacting the sample with at least one chemical entity described herein under conditions that permit detection of Syk activity, detecting a level of Syk activity in the sample, and therefrom determining the presence or absence of Syk in the sample.
  • Also provided is a method for inhibiting B-cell activity comprising contacting cells expressing Syk with at least one chemical entity described herein in an amount sufficient to detectably decrease B-cell activity in vitro.
  • a dash (“-”) that is not between two letters or symbols is used to indicate a point of attachment for a substituent. For example, —CONH 2 is attached through the carbon atom.
  • optionally substituted alkyl encompasses both “alkyl” and “substituted alkyl” as defined below. It will be understood by those skilled in the art, with respect to any group containing one or more substituents, that such groups are not intended to introduce any substitution or substitution patterns that are sterically impractical, synthetically non-feasible and/or inherently unstable.
  • Alkyl encompasses straight chain and branched chain having the indicated number of carbon atoms, usually from 1 to 20 carbon atoms, for example 1 to 8 carbon atoms, such as 1 to 6 carbon atoms.
  • C 1 -C 6 alkyl encompasses both straight and branched chain alkyl of from 1 to 6 carbon atoms.
  • alkyl groups include methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, pentyl, 2-pentyl, isopentyl, neopentyl, hexyl, 2-hexyl, 3-hexyl, 3-methylpentyl, and the like.
  • Alkylene is another subset of alkyl, referring to the same residues as alkyl, but having two points of attachment. Alkylene groups will usually have from 2 to 20 carbon atoms, for example 2 to 8 carbon atoms, such as from 2 to 6 carbon atoms. For example, C 0 alkylene indicates a covalent bond and C 1 alkylene is a methylene group.
  • alkyl residue having a specific number of carbons is named, all geometric isomers having that number of carbons are intended to be encompassed; thus, for example, “butyl” is meant to include n-butyl, sec-butyl, isobutyl and t-butyl; “propyl” includes n-propyl and isopropyl. “Lower alkyl” refers to alkyl groups having 1 to 4 carbons.
  • Alkenyl indicates an unsaturated branched or straight-chain alkyl group having at least one carbon-carbon double bond derived by the removal of one molecule of hydrogen from adjacent carbon atoms of the parent alkyl.
  • the group may be in either the cis or trans configuration about the double bond(s).
  • Typical alkenyl groups include, but are not limited to, ethenyl; propenyls such as prop-1-en-1-yl, prop-1-en-2-yl, prop-2-en-1-yl (allyl), prop-2-en-2-yl; butenyls such as but-1-en-1-yl, but-1-en-2-yl, 2-methyl-prop-1-en-1-yl, but-2-en-1-yl, but-2-en-1-yl, but-2-en-2-yl, buta-1,3-dien-1-yl, buta-1,3-dien-2-yl; and the like.
  • an alkenyl group has from 2 to 20 carbon atoms and in other embodiments, from 2 to 6 carbon atoms.
  • Alkynyl indicates an unsaturated branched or straight-chain alkyl group having at least one carbon-carbon triple bond derived by the removal of two molecules of hydrogen from adjacent carbon atoms of the parent alkyl.
  • Typical alkynyl groups include, but are not limited to, ethynyl; propynyls such as prop-1-yn-1-yl, prop-2-yn-1-yl; butynyls such as but-1-yn-1-yl, but-1-yn-3-yl, but-3-yn-1-yl; and the like.
  • an alkynyl group has from 2 to 20 carbon atoms and in other embodiments, from 3 to 6 carbon atoms.
  • Cycloalkyl indicates a saturated hydrocarbon ring group, having the specified number of carbon atoms, usually from 3 to 7 ring carbon atoms.
  • Examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl as well as bridged and caged saturated ring groups such as norbornane.
  • cycloalkyloxy refers to the group —O-cycloalkyl.
  • alkoxy is meant an alkyl group of the indicated number of carbon atoms attached through an oxygen bridge such as, for example, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, sec-butoxy, tert-butoxy, pentoxy, 2-pentyloxy, isopentoxy, neopentoxy, hexoxy, 2-hexoxy, 3-hexoxy, 3-methylpentoxy, and the like.
  • Alkoxy groups will usually have from 1 to 6 carbon atoms attached through the oxygen bridge. “Lower alkoxy” refers to alkoxy groups having 1 to 4 carbons.
  • Aminocarbonyl encompasses a group of the formula —(C ⁇ O)NR a R b where R a and R b are independently chosen from hydrogen and the optional substituents for “substituted amino” described below.
  • Acyl refers to the groups (alkyl)-C(O)—; (cycloalkyl)-C(O)—; (aryl)-C(O)—; (heteroaryl)-C(O)—; and (heterocycloalkyl)-C(O)—, wherein the group is attached to the parent structure through the carbonyl functionality and wherein alkyl, cycloalkyl, aryl, heteroaryl, and heterocycloalkyl are as described herein.
  • Acyl groups have the indicated number of carbon atoms, with the carbon of the keto group being included in the numbered carbon atoms.
  • a C 2 acyl group is an acetyl group having the formula CH 3 (C ⁇ O)—.
  • alkoxycarbonyl is meant an ester group of the formula (alkoxy)(C ⁇ O)— attached through the carbonyl carbon wherein the alkoxy group has the indicated number of carbon atoms.
  • a C 1 -C 6 alkoxycarbonyl group is an alkoxy group having from 1 to 6 carbon atoms attached through its oxygen to a carbonyl linker.
  • amino is meant the group —NH 2 .
  • aryloxy refers to the group —O-aryl.
  • halo includes fluoro, chloro, bromo, and iodo
  • halogen includes fluorine, chlorine, bromine, and iodine
  • Heteroaryl encompasses:
  • Substituted heteroaryl also includes ring systems substituted with one or more oxide (—O ⁇ ) substituents, such as pyridinyl N-oxides.
  • heteroaryloxy refers to the group —O-heteroaryl.
  • heterocycloalkyl is meant a single aliphatic ring, usually with 3 to 7 ring atoms, containing at least 2 carbon atoms in addition to 1-3 heteroatoms independently selected from oxygen, sulfur, and nitrogen, as well as combinations comprising at least one of the foregoing heteroatoms.
  • Suitable heterocycloalkyl groups include, for example (as numbered from the linkage position assigned priority 1), 2-pyrrolinyl, 2,4-imidazolidinyl, 2,3-pyrazolidinyl, 2-piperidyl, 3-piperidyl, 4-piperdyl, and 2,5-piperzinyl.
  • Morpholinyl groups are also contemplated, including 2-morpholinyl and 3-morpholinyl (numbered wherein the oxygen is assigned priority 1).
  • Substituted heterocycloalkyl also includes ring systems substituted with one or more oxo moieties, such as piperidinyl N-oxide, morpholinyl-N-oxide, 1-oxo-1-thiomorpholinyl and 1,1-dioxo-1-thiomorpholinyl.
  • heterocycloalkyloxy refers to the group —O-heterocylcoalkyl.
  • nitro refers to the group —NO 2 .
  • phosphono refers to the group —PO 3 H 2 .
  • Thiocarbonyl refers to the group —C( ⁇ O)SH.
  • optionally substituted thiocarbonyl includes the following groups: —C( ⁇ O)S-(optionally substituted (C 1 -C 6 )alkyl), —C( ⁇ O)S-(optionally substituted aryl), —C( ⁇ O)S-(optionally substituted heteroaryl), and —C( ⁇ O)S-(optionally substituted heterocycloalkyl).
  • sulfanyl includes the groups: —S-(optionally substituted (C 1 -C 6 )alkyl), —S-(optionally substituted aryl), —S-(optionally substituted heteroaryl), and —S-(optionally substituted heterocycloalkyl).
  • sulfanyl includes the group C 1 -C 6 alkylsulfanyl.
  • sulfinyl includes the groups: —S(O)—H, —S(O)-(optionally substituted (C 1 -C 6 )alkyl), —S(O)-optionally substituted aryl), —S(O)-optionally substituted heteroaryl), —S(O)-(optionally substituted heterocycloalkyl); and —S(O)-(optionally substituted amino).
  • sulfonyl includes the groups: —S(O 2 )—H, —S(O 2 )-(optionally substituted (C 1 -C 6 )alkyl), —S(O 2 )-optionally substituted aryl), —S(O 2 )-optionally substituted heteroaryl), —S(O 2 )-(optionally substituted heterocycloalkyl), —S(O 2 )-(optionally substituted alkoxy), —S(O 2 )-optionally substituted aryloxy), —S(O 2 )-optionally substituted heteroaryloxy), —S(O 2 )-(optionally substituted heterocyclyloxy); and —S(O 2 )-(optionally substituted amino).
  • substituted means that any one or more hydrogens on the designated atom or group is replaced with a selection from the indicated group, provided that the designated atom's normal valence is not exceeded.
  • a substituent is oxo (i.e., ⁇ O) then 2 hydrogens on the atom are replaced.
  • Combinations of substituents and/or variables are permissible only if such combinations result in stable compounds or useful synthetic intermediates.
  • a stable compound or stable structure is meant to imply a compound that is sufficiently robust to survive isolation from a reaction mixture, and subsequent formulation as an agent having at least practical utility.
  • substituents are named into the core structure. For example, it is to be understood that when (cycloalkyl)alkyl is listed as a possible substituent, the point of attachment of this substituent to the core structure is in the alkyl portion.
  • substituted alkyl, cycloalkyl, aryl, heterocycloalkyl, and heteroaryl refer respectively to alkyl, cycloalkyl, aryl, heterocycloalkyl, and heteroaryl wherein one or more (such as up to 5, for example, up to 3) hydrogen atoms are replaced by a substituent independently chosen from:
  • substituted acyl refers to the groups (substituted alkyl)-C(O)—; (substituted cycloalkyl)-C(O)—; (substituted aryl)-C(O)—; (substituted heteroaryl)-C(O)—; and (substituted heterocycloalkyl)-C(O)—, wherein the group is attached to the parent structure through the carbonyl functionality and wherein substituted alkyl, cycloalkyl, aryl, heteroaryl, and heterocycloalkyl, refer respectively to alkyl, cycloalkyl, aryl, heteroaryl, and heterocycloalkyl wherein one or more (such as up to 5, for example, up to 3) hydrogen atoms are replaced by a substituent independently chosen from:
  • substituted alkoxy refers to alkoxy wherein the alkyl constituent is substituted (i.e., —O-(substituted alkyl)) wherein “substituted alkyl” refers to alkyl wherein one or more (such as up to 5, for example, up to 3) hydrogen atoms are replaced by a substituent independently chosen from:
  • substituted alkoxycarbonyl refers to the group (substituted alkyl)-O—C(O)— wherein the group is attached to the parent structure through the carbonyl functionality and wherein substituted refers to alkyl wherein one or more (such as up to 5, for example, up to 3) hydrogen atoms are replaced by a substituent independently chosen from:
  • substituted aryloxy refers to aryloxy wherein the aryl constituent is substituted (i.e., —O-(substituted aryl)) wherein “substituted aryl” refers to aryl wherein one or more (such as up to 5, for example, up to 3) hydrogen atoms are replaced by a substituent independently chosen from:
  • substituted heteroaryloxy refers to heteroaryloxy wherein the aryl constituent is substituted (i.e., —O-(substituted heteroaryl)) wherein “substituted heteroaryl” refers to aryl wherein one or more (such as up to 5, for example, up to 3) hydrogen atoms are replaced by a substituent independently chosen from:
  • substituted cycloalkyloxy refers to cycloalkyloxy wherein the cycloalkyl constituent is substituted (i.e., —O-(substituted cycloalkyl)) wherein “substituted cycloalkyl” refers to a cycloalkyl wherein one or more hydrogen atoms are replaced by a substituent independently chosen from:
  • substituted heterocycloalkyloxy refers to heterocycloalkyloxy wherein the alkyl constituent is substituted (i.e., —O-(substituted heterocycloalkyl)) wherein “substituted heterocycloalkyl” refers to a heterocycloalkyloxy wherein one or more hydrogen atoms are replaced by a substituent independently chosen from:
  • substituted amino refers to the group —NHR d or —NR d R d where each R d is independently chosen from: hydroxy, optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted acyl, aminocarbonyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocycloalkyl, alkoxycarbonyl, sulfinyl and sulfonyl, provided that only one R d may be hydroxyl, and wherein substituted alkyl, cycloalkyl, aryl, heterocycloalkyl, and heteroaryl refer respectively to alkyl, cycloalkyl, aryl, heterocycloalkyl, and heteroaryl wherein one or more (such as up to 5, for example, up to 3) hydrogen atoms are replaced by a substituent independently chosen from:
  • substituted amino also refers to N-oxides of the groups —NHR d , and NR d R d each as described above.
  • N-oxides can be prepared by treatment of the corresponding amino group with, for example, hydrogen peroxide or m-chloroperoxybenzoic acid. The person skilled in the art is familiar with reaction conditions for carrying out the N-oxidation.
  • Compounds described herein include, but are not limited to, their optical isomers, racemates, and other mixtures thereof.
  • the single enantiomers or diastereomers i.e., optically active forms
  • Resolution of the racemates can be accomplished, for example, by conventional methods such as crystallization in the presence of a resolving agent, or chromatography, using, for example a chiral high-pressure liquid chromatography (HPLC) column.
  • HPLC high-pressure liquid chromatography
  • such compounds include Z- and E-forms (or cis- and trans-forms) of compounds with carbon-carbon double bonds.
  • chemical entities include all tautomeric forms of the compound.
  • Such compounds also include crystal forms including polymorphs and clathrates.
  • Chemical entities include, but are not limited to compounds described herein and all pharmaceutically acceptable forms thereof.
  • Pharmaceutically acceptable forms of the compounds recited herein include pharmaceutically acceptable salts, solvates, chelates, non-covalent complexes, prodrugs, and mixtures thereof.
  • the compounds described herein are in the form of pharmaceutically acceptable salts.
  • the terms “chemical entity” and “chemical entities” also encompass pharmaceutically acceptable salts, solvates, chelates, non-covalent complexes, prodrugs, and mixtures.
  • “Pharmaceutically acceptable salts” include, but are not limited to salts with inorganic acids, such as hydrochlorate, phosphate, diphosphate, hydrobromate, sulfate, sulfinate, nitrate, and like salts; as well as salts with an organic acid, such as malate, maleate, fumarate, tartrate, succinate, citrate, acetate, lactate, methanesulfonate, p-toluenesulfonate, 2-hydroxyethylsulfonate, benzoate, salicylate, stearate, and alkanoate such as acetate, HOOC—(CH 2 ) n —COOH where n is 0-4, and like salts.
  • pharmaceutically acceptable cations include, but are not limited to sodium, potassium, calcium, aluminum, lithium, and ammonium.
  • the free base can be obtained by basifying a solution of the acid salt.
  • an addition salt particularly a pharmaceutically acceptable addition salt, may be produced by dissolving the free base in a suitable organic solvent and treating the solution with an acid, in accordance with conventional procedures for preparing acid addition salts from base compounds.
  • Those skilled in the art will recognize various synthetic methodologies that may be used to prepare non-toxic pharmaceutically acceptable addition salts.
  • prodrugs also fall within the scope of chemical entities, for example ester or amide derivatives of the compounds described herein.
  • the term “prodrugs” includes any compounds that become compounds described herein when administered to a patient, e.g., upon metabolic processing of the prodrug.
  • Examples of prodrugs include, but are not limited to, acetate, formate, and benzoate and like derivatives of functional groups (such as alcohol or amine groups) in the compounds of described herein.
  • solvate refers to the chemical entity formed by the interaction of a solvent and a compound. Suitable solvates are pharmaceutically acceptable solvates, such as hydrates, including monohydrates and hemi-hydrates.
  • chelate refers to the chemical entity formed by the coordination of a compound to a metal ion at two (or more) points.
  • non-covalent complex refers to the chemical entity formed by the interaction of a compound and another molecule wherein a covalent bond is not formed between the compound and the molecule.
  • complexation can occur through van der Waals interactions, hydrogen bonding, and electrostatic interactions (also called ionic bonding).
  • hydrogen bond refers to a form of association between an electronegative atom (also known as a hydrogen bond acceptor) and a hydrogen atom attached to a second, relatively electronegative atom (also known as a hydrogen bond donor).
  • Suitable hydrogen bond donor and acceptors are well understood in medicinal chemistry (G. C. Pimentel and A. L. McClellan, The Hydrogen Bond, Freeman, San Francisco, 1960; R. Taylor and O. Kennard, “Hydrogen Bond Geometry in Organic Crystals”, Accounts of Chemical Research, 17, pp. 320-326 (1984)).
  • Haldrogen bond acceptor refers to a group comprising an oxygen or nitrogen, especially an oxygen or nitrogen that is sp 2 -hybridized, an ether oxygen, or the oxygen of a sulfoxide or N-oxide.
  • hydrogen bond donor refers to an oxygen, nitrogen, or heteroaromatic carbon that bears a hydrogen group containing a ring nitrogen or a heteroaryl group containing a ring nitrogen.
  • group As used herein the terms “group”, “radical” or “fragment” are synonymous and are intended to indicate functional groups or fragments of molecules attachable to a bond or other fragments of molecules.
  • an “active agent” is used to indicate a chemical entity which has biological activity.
  • an “active agent” is a compound having pharmaceutical utility.
  • an active agent may be an anti-cancer therapeutic.
  • a therapeutically effective amount of a chemical entity described herein means an amount effective, when administered to a human or non-human patient, to provide a therapeutic benefit such as amelioration of symptoms, slowing of disease progression, or prevention of disease e.g., a therapeutically effective amount may be an amount sufficient to decrease the symptoms of a disease responsive to inhibition of Syk activity.
  • a therapeutically effective amount is an amount sufficient to reduce cancer symptoms, the symptoms of an allergic disorder, the symptoms of an autoimmune and/or inflammatory disease, or the symptoms of an acute inflammatory reaction.
  • a therapeutically effective amount is an amount sufficient to decrease the number of detectable cancerous cells in an organism, detectably slow, or stop the growth of a cancerous tumor.
  • a therapeutically effective amount is an amount sufficient to shrink a cancerous tumor. In some circumstances a patient suffering from cancer may not present symptoms of being affected.
  • a therapeutically effective amount of a chemical entity is an amount sufficient to prevent a significant increase or significantly reduce the detectable level of cancerous cells or cancer markers in the patient's blood, serum, or tissues.
  • a therapeutically effective amount may also be an amount sufficient, when administered to a patient, to detectably slow progression of the disease, or prevent the patient to whom the chemical entity is given from presenting symptoms of the allergic disorders and/or autoimmune and/or inflammatory disease, and/or acute inflammatory response.
  • a therapeutically effective amount may also be an amount sufficient to produce a detectable decrease in the amount of a marker protein or cell type in the patient's blood or serum.
  • a therapeutically effective amount is an amount of a chemical entity described herein sufficient to significantly decrease the activity of B-cells.
  • a therapeutically effective amount is an amount of a chemical entity described herein sufficient to significantly decrease the number of B-cells.
  • a therapeutically effective amount is an amount of a chemical entity described herein sufficient to decrease the level of anti-acetylcholine receptor antibody in a patient's blood with the disease myasthenia gravis.
  • inhibitortion indicates a significant decrease in the baseline activity of a biological activity or process.
  • “Inhibition of Syk activity” refers to a decrease in Syk activity as a direct or indirect response to the presence of at least one chemical entity described herein, relative to the activity of Syk in the absence of the at least one chemical entity.
  • the decrease in activity may be due to the direct interaction of the compound with Syk, or due to the interaction of the chemical entity(ies) described herein with one or more other factors that in turn affect Syk activity.
  • the presence of the chemical entity(ies) may decrease Syk activity by directly binding to the Syk, by causing (directly or indirectly) another factor to decrease Syk activity, or by (directly or indirectly) decreasing the amount of Syk present in the cell or organism.
  • Inhibition of Syk activity also refers to observable inhibition of Syk activity in a standard biochemical assay for Syk activity, such as the ATP hydrolysis assay described below.
  • the chemical entity described herein has an IC 50 value less than or equal to 1 micromolar. In some embodiments, the chemical entity has an IC 50 value less than or equal to less than 100 nanomolar. In some embodiments, the chemical entity has an IC 50 value less than or equal to 10 nanomolar.
  • “Inhibition of B-cell activity” refers to a decrease in B-cell activity as a direct or indirect response to the presence of at least one chemical entity described herein, relative to the activity of B-cells in the absence of the at least one chemical entity.
  • the decrease in activity may be due to the direct interaction of the compound with Syk or with one or more other factors that in turn affect B-cell activity.
  • Inhibition of B-cell activity also refers to observable inhibition of CD86 expression in a standard assay such as the assay described below.
  • the chemical entity described herein has an IC 50 value less than or equal to 10 micromolar. In some embodiments, the chemical entity has an IC 50 value less than or equal to less than 1 micromolar. In some embodiments, the chemical entity has an IC 50 value less than or equal to 500 nanomolar.
  • B cell activity also includes activation, redistribution, reorganization, or capping of one or more various B cell membrane receptors, or membrane-bound immunoglobulins, e.g, IgM, IgG, and IgD. Most B cells also have membrane receptors for Fc portion of IgG in the form of either antigen-antibody complexes or aggregated IgG. B cells also carry membrane receptors for the activated components of complement, e.g., C3b, C3d, C4, and C1q. These various membrane receptors and membrane-bound immunoglobulins have membrane mobility and can undergo redistribution and capping that can initiate signal transduction.
  • B cell activity also includes the synthesis or production of antibodies or immunoglobulins.
  • Immunoglobulins are synthesized by the B cell series and have common structural features and structural units. Five immunoglobulin classes, i.e., IgG, IgA, IgM, IgD, and IgE, are recognized on the basis of structural differences of their heavy chains including the amino acid sequence and length of the polypeptide chain.
  • Antibodies to a given antigen may be detected in all or several classes of immunoglobulins or may be restricted to a single class or subclass of immunoglobulin.
  • Autoantibodies or autoimmune antibodies may likewise belong to one or several classes of immunoglobulins. For example, rheumatoid factors (antibodies to IgG) are most often recognized as an IgM immunoglobulin, but can also consist of IgG or IgA.
  • B cell activity also is intended to include a series of events leading to B cell clonal expansion (proliferation) from precursor B lymphocytes and differentiation into antibody-synthesizing plasma cells which takes place in conjunction with antigen-binding and with cytokine signals from other cells.
  • “Inhibition of B-cell proliferation” refers to inhibition of proliferation of abnormal B-cells, such as cancerous B-cells, e.g. lymphoma B-cells and/or inhibition of normal, non-diseased B-cells.
  • the term “inhibition of B-cell proliferation” indicates any significant decrease in the number of B-cells, either in vitro or in vivo. Thus an inhibition of B-cell proliferation in vitro would be any significant decrease in the number of B-cells in an in vitro sample contacted with at least one chemical entity described herein as compared to a matched sample not contacted with the chemical entity(ies).
  • Inhibition of B-cell proliferation also refers to observable inhibition of B-cell proliferation in a standard thymidine incorporation assay for B-cell proliferation, such as the assay described herein.
  • the chemical entity has an IC 50 value less than or equal to 10 micromolar. In some embodiments, the chemical entity has an IC 50 value less than or equal to less than 1 micromolar. In some embodiments, the chemical entity has an IC 50 value less than or equal to 500 nanomolar.
  • Allergic disorder refers to acquired hypersensitivity to a substance (allergen). Allergic conditions include eczema, allergic rhinitis or coryza, hay fever, bronchial asthma, urticaria (hives) and food allergies, and other atopic conditions.
  • Asthma refers to a disorder of the respiratory system characterized by inflammation, narrowing of the airways and increased reactivity of the airways to inhaled agents. Asthma is frequently, although not exclusively associated with atopic or allergic symptoms.
  • significant is meant any detectable change that is statistically significant in a standard parametric test of statistical significance such as Student's T-test, where p ⁇ 0.05.
  • a “disease responsive to inhibition of Syk activity” is a disease in which inhibiting Syk kinase provides a therapeutic benefit such as an amelioration of symptoms, decrease in disease progression, prevention or delay of disease onset, or inhibition of aberrant activity of certain cell-types (monocytes, B-cells, and mast cells).
  • Treatment or treating means any treatment of a disease in a patient, including:
  • Patient refers to an animal, such as a mammal, that has been or will be the object of treatment, observation or experiment. The methods described herein may be useful in both human therapy and veterinary applications.
  • the patient is a mammal; in some embodiments the patient is human; and in some embodiments the patient is chosen from cats and dogs.
  • Q is SO 2 . In some embodiments, Q is C(O).
  • W is optionally substituted aryl. In some embodiments, W is optionally substituted heteroaryl.
  • A is selected from halo, optionally substituted lower alkyl, and hydrogen. In some embodiments, A is selected from halo, lower alkyl, and hydrogen. In some embodiments, A is hydrogen.
  • X is CH. In some embodiments, X is N.
  • Y is selected from optionally substituted lower alkyl and hydrogen. In some embodiments, Y is selected from lower alkyl and hydrogen. In some embodiments, Y is hydrogen.
  • Y and Z are taken together with the nitrogen to which they are bound to form a 5- to 13-membered nitrogen-containing ring. In some embodiments, Y and Z are taken together with the nitrogen to which they are bound to form a 5- to 13-membered nitrogen-containing ring selected from optionally substituted piperazinyl, optionally substituted piperidinyl and optionally substituted diazepanyl. In some embodiments, Z taken together with Y, and the nitrogen to which they are bound, form an optionally substituted 5- to 13-membered nitrogen-containing ring which also contains an additional oxygen heteroatom. In some embodiments, Z taken together with Y, and the nitrogen to which they are bound, form an optionally substituted morpholinyl ring.
  • Z is selected from hydrogen, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted cycloalkyl and optionally substituted heterocycloakyl. In some embodiments, Z is selected from optionally substituted phenyl, optionally substituted piperidinyl, optionally substituted benzodiazolyl, optionally substituted pyridinyl and optionally substituted cyclohexyl.
  • Z is selected from hydrogen and lower alkyl that is optionally substituted with a group chosen from amino, (lower alkyl)amino, di-(lower alkyl)amino, hydroxy, and lower alkoxy. In some embodiments, Z is selected from hydrogen.
  • Z is represented by the following structure:
  • Y and Z are both hydrogen.
  • V is C. In some embodiments, V is N.
  • R 6 , R 7 , R 8 , R 9 and R 10 are each independently selected from optionally substituted C 1 -C 6 alkoxy, optionally substituted C 1 -C 6 alkyl, optionally substituted C 1 -C 6 alkylcarbonyl, optionally substituted aminocarbonyl, optionally substituted aryl, carboxy, cyano, optionally substituted C 1 -C 6 alkoxycarbonyl, halo, optionally substituted heteroaryl, hydrogen, hydroxy, and sulfonyl.
  • R 6 , R 7 , R 9 and R 10 are each hydrogen.
  • R 8 is selected from optionally substituted C 1 -C 6 alkyl, optionally substituted C 1 -C 6 alkoxy, optionally substituted C 1 -C 6 alkoxycarbonyl, optionally substituted aminocarbonyl, carboxy, cyano, halo, optionally substituted heteroaryl, optionally substituted heterocycloalkyl, hydroxyl and sulfonyl.
  • R 8 is carboxy.
  • R 8 is —C(O)—NH(lower alkyl).
  • R 1 , R 2 , R 3 , R 4 and R 5 are each independently selected from optionally substituted C 1 -C 6 alkoxy, optionally substituted C 1 -C 6 alkyl, optionally substituted aminocarbonyl, optionally substituted heterocycloalkyl and hydrogen. In some embodiments, R 1 , R 2 and R 5 are hydrogen. In some embodiments, R 3 and R 4 are each independently selected from optionally substituted C 1 -C 6 alkoxy, optionally substituted C 1 -C 6 alkyl, and optionally substituted heterocycloalkyl. In some embodiments, R 3 and R 4 are both independently optionally substituted C 1 -C 6 alkoxy. In some embodiments, R 3 and R 4 are both methoxy.
  • the chemical entities can be administered alone, as mixtures, or in combination with other active agents.
  • Step 1 a solution of a compound of Formula 101 in a polar solvent such as dichloromethane is added in excess (such as about 1.1 equivalents) to a compound of Formula 102, where R 12 is a leaving group such as chloride.
  • a tertiary amine such as N,N-diisopropylethylamine is added to the mixture in excess (such as about 3.3 equivalents). The mixture is stirred at room temperature for about 13 h.
  • the product, a compound of Formula 103 is isolated and optionally purified.
  • Step 2 a 1:1 ratio of a compound of Formula 103 and a compound of Formula 204 (where R 13 is a halide such as Br) are taken up in an aqueous solution of base (such as 1M sodium carbonate) and an inert solvent such as 1,4-dioxane.
  • base such as 1M sodium carbonate
  • inert solvent such as 1,4-dioxane.
  • the reaction mixture is sparged with nitrogen and stirred for 5 min.
  • the resulting mixture is treated with about 0.2 equivalent of tetrakis(triphenylphosphine)palladium(0) and stirred to reflux for 2.5 h.
  • the resulting product, a compound of Formula 205 is isolated and optionally purified.
  • Step 1 a mixture of the salt of Formula 301 (such as about 0.7 equivalent), a compound of Formula 101 (such as about 1.0 equivalent), benzotriazol-1-yloxy-tris(dimethylamino)-phosphonium hexafluorophosphate (such as about 1.0 equivalent) and 4-methylmorpholine (such as about 3.3 equivalents) in an inert solvent such as DMF are stirred at room temperature for 18 h. The mixture is then stirred at 45° C. for 6 h. Then, methyl 4-amino-3-methylbenzoate (such as about 1.0 equivalent) is added and the resulting mixture is stirred at room temperature for an additional 18 h.
  • an inert solvent such as DMF
  • benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate such as about 0.7 equivalent
  • benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate such as about 0.7 equivalent
  • Step 1 a compound of Formula 205 in an aqueous mixture of MeOH is treated with HCl (such as a 2N solution (pH ⁇ 2)) and sonicated for approximately 45 min. Any solids that form are treated with a small volume of 2N HCl and Et 2 O.
  • HCl such as a 2N solution (pH ⁇ 2)
  • Any solids that form are treated with a small volume of 2N HCl and Et 2 O.
  • the resulting product, a salt of Formula 401 is optionally purified by trituration with acetonitrile.
  • a method of treating a patient for example, a mammal, such as a human, having a disease responsive to inhibition of Syk activity, comprising administrating to the patient having such a disease, an effective amount of at least one chemical entity described herein.
  • the chemical entities described herein may also inhibit other kinases, such that disease, disease symptoms, and conditions associated with these kinases is also treated.
  • Methods of treatment also include inhibiting Syk activity and/or inhibiting B-cell activity, by inhibiting ATP binding or hydrolysis by Syk or by some other mechanism, in vivo, in a patient suffering from a disease responsive to inhibition of Syk activity, by administering an effective concentration of at least one chemical entity chosen described herein.
  • An effective concentration would be that concentration sufficient to inhibit Syk activity in vitro.
  • An effective concentration may be ascertained experimentally, for example by assaying blood concentration of the chemical entity, or theoretically, by calculating bioavailability.
  • the condition responsive to inhibition of Syk activity and/or B-cell activity is cancer, an allergic disorder and/or an autoimmune and/or inflammatory disease, and/or an acute inflammatory reaction.
  • the conditions and diseases that can be affected using chemical entities described herein include, but are not limited to: allergic disorders, including but not limited to eczema, allergic rhinitis or coryza, hay fever, bronchial asthma, urticaria (hives) and food allergies, and other atopic conditions; autoimmune and/or inflammatory diseases, including but not limited to psoriasis, Crohn's disease, irritable bowel syndrome, Sjogren's disease, tissue graft rejection, and hyperacute rejection of transplanted organs, asthma, systemic lupus erythematosus (and associated glomerulonephritis), dermatomyositis, multiple sclerosis, scleroderma, vasculitis (ANCA-associated and other vasculitides), autoimmune hemolytic and thrombocytopenic states, Goodpasture's syndrome (and associated glomerulonephritis and pulmonary hemorrhage), atherosclerosis, rhe
  • Syk is a known inhibitor of apoptosis in lymphoma B-cells. Defective apoptosis contributes to the pathogenesis and drug resistance of human leukemias and lymphomas.
  • a method of promoting or inducing apoptosis in cells expressing Syk comprising contacting the cell with at least one chemical entity described herein.
  • a method of treating cancer, an allergic disorder and/or an autoimmune and/or inflammatory disease, and/or an acute inflammatory reaction comprises administering to a patient in need thereof an effective amount of at least one chemical entity described herein, together with a second active agent, which can be useful for treating a cancer, an allergic disorder and/or an autoimmune and/or inflammatory disease, and/or an acute inflammatory reaction.
  • the second agent may be an anti-inflammatory agent.
  • Treatment with the second active agent may be prior to, concomitant with, or following treatment with at least one chemical entity described herein.
  • at least one chemical entity described herein is combined with another active agent in a single dosage form.
  • Suitable antitumor therapeutics that may be used in combination with at least one chemical entity described herein include, but are not limited to, chemotherapeutic agents, for example mitomycin C, carboplatin, taxol, cisplatin, paclitaxel, etoposide, doxorubicin, or a combination comprising at least one of the foregoing chemotherapeutic agents. Radiotherapeutic antitumor agents may also be used, alone or in combination with chemotherapeutic agents.
  • Chemical entities described herein can be useful as chemosensitizing agents, and, thus, can be useful in combination with other chemotherapeutic drugs, in particular, drugs that induce apoptosis.
  • a method for increasing sensitivity of cancer cells to chemotherapy comprising administering to a patient undergoing chemotherapy a chemotherapeutic agent together with at least one chemical entity described herein in an amount sufficient to increase the sensitivity of cancer cells to the chemotherapeutic agent is also provided herein.
  • chemotherapeutic drugs examples include topoisomerase I inhibitors (camptothesin or topotecan), topoisomerase II inhibitors (e.g. daunomycin and etoposide), alkylating agents (e.g. cyclophosphamide, melphalan and BCNU), tubulin directed agents (e.g. taxol and vinblastine), and biological agents (e.g. antibodies such as anti CD20 antibody, IDEC 8, immunotoxins, and cytokines).
  • topoisomerase I inhibitors camptothesin or topotecan
  • topoisomerase II inhibitors e.g. daunomycin and etoposide
  • alkylating agents e.g. cyclophosphamide, melphalan and BCNU
  • tubulin directed agents e.g. taxol and vinblastine
  • biological agents e.g. antibodies such as anti CD20 antibody, IDEC 8, immunotoxins, and cytokines.
  • the chemical entities described herein are used in combination with Rituxan® (Rituximab) or other agents that work by selectively depleting CD20+ B-cells.
  • Anti-inflammatory agents include but are not limited to NSAIDs, non-specific and COX-2 specific cyclooxgenase enzyme inhibitors, gold compounds, corticosteroids, methotrexate, tumor necrosis factor receptor (TNF) receptors antagonists, immunosuppressants and methotrexate.
  • NSAIDs include, but are not limited to ibuprofen, flurbiprofen, naproxen and naproxen sodium, diclofenac, combinations of diclofenac sodium and misoprostol, sulindac, oxaprozin, diflunisal, piroxicam, indomethacin, etodolac, fenoprofen calcium, ketoprofen, sodium nabumetone, sulfasalazine, tolmetin sodium, and hydroxychloroquine.
  • NSAIDs also include COX-2 specific inhibitors (i.e., a compound that inhibits COX-2 with an IC 50 that is at least 50-fold lower than the IC 50 for COX-1) such as celecoxib, valdecoxib, lumiracoxib, etoricoxib and/or rofecoxib.
  • COX-2 specific inhibitors i.e., a compound that inhibits COX-2 with an IC 50 that is at least 50-fold lower than the IC 50 for COX-1
  • celecoxib valdecoxib
  • lumiracoxib etoricoxib
  • etoricoxib etoricoxib
  • rofecoxib rofecoxib
  • the anti-inflammatory agent is a salicylate.
  • Salicylates include but are not limited to acetylsalicylic acid or aspirin, sodium salicylate, and choline and magnesium salicylates.
  • the anti-inflammatory agent may also be a corticosteroid.
  • the corticosteroid may be chosen from cortisone, dexamethasone, methylprednisolone, prednisolone, prednisolone sodium phosphate, and prednisone.
  • the anti-inflammatory therapeutic agent is a gold compound such as gold sodium thiomalate or auranofin.
  • the anti-inflammatory agent is a metabolic inhibitor such as a dihydrofolate reductase inhibitor, such as methotrexate or a dihydroorotate dehydrogenase inhibitor, such as leflunomide.
  • a metabolic inhibitor such as a dihydrofolate reductase inhibitor, such as methotrexate or a dihydroorotate dehydrogenase inhibitor, such as leflunomide.
  • At least one anti-inflammatory compound is an anti-C5 monoclonal antibody (such as eculizumab or pexelizumab), a TNF antagonist, such as entanercept, or infliximab, which is an anti-TNF alpha monoclonal antibody are used.
  • an anti-C5 monoclonal antibody such as eculizumab or pexelizumab
  • a TNF antagonist such as entanercept, or infliximab, which is an anti-TNF alpha monoclonal antibody
  • At least one active agent is an immunosuppressant compound such as methotrexate, leflunomide, cyclosporine, tacrolimus, azathioprine, or mycophenolate mofetil are used.
  • Dosage levels of the order for example, of from 0.1 mg to 140 mg per kilogram of body weight per day can be useful in the treatment of the above-indicated conditions (0.5 mg to 7 g per patient per day).
  • the amount of active ingredient that may be combined with the vehicle to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. Dosage unit forms will generally contain from 1 mg to 500 mg of an active ingredient.
  • Frequency of dosage may also vary depending on the compound used and the particular disease treated. In some embodiments, for example, for the treatment of an allergic disorder and/or autoimmune and/or inflammatory disease, a dosage regimen of 4 times daily or less is used. In some embodiments, a dosage regimen of 1 or 2 times daily is used. It will be understood, however, that the specific dose level for any particular patient will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, and rate of excretion, drug combination and the severity of the particular disease in the patient undergoing therapy.
  • a labeled form of a chemical entity described herein can be used as a diagnostic for identifying and/or obtaining compounds that have the function of modulating an activity of a kinase as described herein.
  • the chemical entities described herein may additionally be used for validating, optimizing, and standardizing bioassays.
  • label herein is meant that the compound is either directly or indirectly labeled with a label which provides a detectable signal, e.g., radioisotope, fluorescent tag, enzyme, antibodies, particles such as magnetic particles, chemiluminescent tag, or specific binding molecules, etc.
  • Specific binding molecules include pairs, such as biotin and streptavidin, digoxin and antidigoxin etc.
  • the complementary member would normally be labeled with a molecule which provides for detection, in accordance with known procedures, as outlined above.
  • the label can directly or indirectly provide a detectable signal.
  • DME dimethyl ether
  • DMEM Dulbecco's modified Eagle's medium
  • DMF N,N-dimethylformamide
  • DMSO dimethylsulfoxide
  • Et 2 O diethylether
  • mM millimolar
  • the resulting mixture was treated with tetrakis(triphenylphosphine)palladium(0) (1.20 g, 1.04 mmol) and stirred at reflux for 2.5 h. After this time, the reaction was cooled to ambient temperature, diluted with ethyl acetate (100 mL) and washed with brine (100 mL). The aqueous layer was extracted with ethyl acetate (100 mL) and the combined organic layers were dried over sodium sulfate, filtered and the filtrate was concentrated under reduced pressure.
  • Electrospray MS is performed on a MICROMASS LCT equipped with a LockSpray source for accurate mass measurements. Spectra are acquired in positive ion mode from 100-1000 Da at an acquisition rate of 1 spectrum/0.9 s with a 0.1 s interscan delay. The instrument is tuned for a resolution of 5000 (FWHM). Every 5 th scan is taken from the reference position of the Lockspray source. Leucine enkephalin (556.2771 [M+H] + ) is used as the reference or lock mass.
  • a master mix minus Syk enzyme is prepared containing 1 ⁇ Cell Signaling kinase buffer (25 mM Tris-HCl, pH 7.5, 5 mM beta-glycerophosphate, 2 mM dithiothreitol, 0.1 mM Na 3 VO 4 , 10 mM MgCl 2 ), 0.5 ⁇ M Promega PTK Biotinylated peptide substrate 1, 0.01% casein, 0.01% Triton-X100, and 0.25% glycerol.
  • 1 ⁇ Cell Signaling kinase buffer 25 mM Tris-HCl, pH 7.5, 5 mM beta-glycerophosphate, 2 mM dithiothreitol, 0.1 mM Na 3 VO 4 , 10 mM MgCl 2
  • 0.5 ⁇ M Promega PTK Biotinylated peptide substrate 1, 0.01% casein, 0.01% Triton-X100, and 0.25% gly
  • a master mix plus Syk enzyme is prepared containing 1 ⁇ Cell Signaling kinase buffer, 0.5 ⁇ M PTK Biotinylated peptide substrate 1, 0.01% casein, 0.01% Triton-X100, 0.25% glycerol and 0.4 ng/well Syk enzyme.
  • Syk enzyme is purchased from Cell Signaling Technologies, expressed in baculovirus and is an N-terminally GST-tagged full length human wildtype Syk.
  • the Syk nucleotide sequence accession number is NM — 003177, and accession number for the encoded Syk enzyme is NP — 003168.
  • the Syk protein was purified in one step using glutathione-agarose.
  • a solution of 200 ⁇ M ATP is prepared in water and adjusted to pH7.4 with 1N NaOH.
  • a quantity of 1.25 ⁇ L of compound in 5% DMSO is transferred to a 96-well 1 ⁇ 2 area Costar polystyrene plate.
  • Compounds are tested singly and with an 11-point dose-responsive curve (starting concentration is 10-1 ⁇ M; 1:2 dilution).
  • a quantity of 18.75 ⁇ L of master mix minus enzyme (as a negative control) and master mix plus enzyme is transferred to appropriate wells in 96-well 1 ⁇ 2 area costar polystyrene plate.
  • Ramos cells are serum starved at 2 ⁇ 10 6 cells/ml in serum-free RPMI for 1 hour in an upright T175 Falcon TC flask. Cells are centrifuged (1100 rpm ⁇ 5 mM) and incubated at a density of 0.5 ⁇ 10 7 cells/ml in the presence of test compound or DMSO controls for 1 hr at 37° C. Cells are then stimulated by incubating with 10 ⁇ g/ml anti-human IgM F(ab) 2 for 5 minutes at 37° C. Cells are pelleted, lysed in 40 ul cell lysis buffer, and mixed with Invitrogen SDS-PAGE loading buffer.
  • 20 ul of cell lysate for each sample are subject to SDS-PAGE and western blotting with anti-phosphoBLNK(Tyr96) antibody (Cell Signaling Technology #3601) to assess Syk activity and anti-Syk antibody (BD Transduction Labs #611116) to control for total protein load in each lysate.
  • the images are detected using fluorescent secondary detection systems and the LiCor Odyssey instrument.
  • B-cells are purified from spleens of 8-16 week old Balb/c mice using a B-cell isolation kit (Miltenyi Biotech, Cat #130-090-862). Test compounds are diluted in 0.25% DMSO and incubated with 2.5 ⁇ 10 5 purified mouse splenic B-cells for 30 min prior to addition of 10 ⁇ g/ml of an anti-mouse IgM antibody (Southern Biotechnology Associates Cat #1022-01) in a final volume of 100 ⁇ l.
  • T cells are purified from spleens of 8-16 week old Balb/c mice using a Pan T cell isolation kit (Miltenyi Biotech, Cat #130-090-861). Test compounds are diluted in 0.25% DMSO and incubated with 2.5 ⁇ 10 5 purified mouse splenic T cells in a final volume of 100 ⁇ l in flat clear bottom plates precoated for 90 min at 37° C. with 10 ⁇ g/ml each of anti-CD3 (BD #553057) and anti-CD28 (BD #553294) antibodies.
  • Total mouse splenocytes are purified from spleens of 8-16 week old Balb/c mice by red blood cell lysis (BD Pharmingen #555899). Testing compounds are diluted to 0.5% DMSO and incubated with 1.25 ⁇ 10 6 splenocytes in a final volume of 200 ⁇ l in flat clear bottom plates (Falcon 353072) for 60 min at 37° C. Cells are then stimulated with the addition of 15 ⁇ g/ml IgM (Jackson ImmunoResearch 115-006-020), and incubated for 16 hr at 37° C., 5% CO 2 .
  • BMMC bone-marrow derived mouse mast cell
  • Bone-marrow derived mast cells were cultured for >4 weeks with IL-3 (10 ng/ml) and SCF (10 ng/ml). The cells were determined to be >90% cKit + /FceRI + by FACS analysis at the time of use.
  • Cells (6 ⁇ 10 7 cells/50 ml) were serum-starved in a T150 tissue culture flask for 16 h in the absence of IL-3 and SCF containing IgE anti-DNP at 1 ug/ml. Overnight sensitized cells are washed twice in Tyrodes buffer and resuspended to 5 ⁇ 10 6 cells/ml.
  • 5 ⁇ 10 5 cells (100 ul) are plated in a 96 well microtiter plate (Falcon 353072) and test compounds are serially diluted to a final concentration 0.25% DMSO in the plate for 1 hr at 37° C., 5% CO 2 .
  • Wells are treated with a DNP-BSA antigen challenge (50 ng/ml) and incubated for and additional 30 min at 37° C.
  • Supernatants are assayed for hexosamimidase release versus control wells.
  • Cell pellets are simultaneously lysed and assessed for total hexosamimidase release to calculate specific release. Dose-response curves are generated using 4-parameter logistical fit and IC50s calculated.
  • PCA Passive Cutaneous Anaphylaxis
  • This solution or a further dilution in saline is diluted 1:1 with 2% Evan's Blue in sterile saline that has been filtered though 0.02 um filter and refiltered prior to injection.
  • a final solution of 0.5 mg/ml of DNP-BSA in 1% Evans blue can be used.
  • -Evan's blue dye A 2% stock in saline will be sterile filtered and diluted 1:1 with DNP-BSA saline solution for final concentration of 1% for injection.
  • mice On d0, animals, anesthetized with isofluorine, are passively sensitized by intradermal injections of IgE anti-DNP using a 29-gauge insulin syringe.
  • the right ear receives 10 ul intradermal injection of IgE anti-DNP while the left ear receives PBS.
  • antigen challenge is administered by tail i.v. injection of DNP-BSA in 200 ul of 1% Evans blue dye solution in saline. Tails are immersed in warm water prior to iv injection to improve success. 3) 30 minutes to 2 hr prior to this antigen challenge, drug is delivered sc or po in 10% EtOH/20% cremaphor/70% saline. 4) Animals are sacrifice by CO 2 inhalation 30-60 min post antigen challenge and ears are removed for extraction of Evan's blue dye in 500 ul of formamide overnight at 65° C. 5) Blood is obtained by cardiac puncture just prior to final cervical dislocation and processed for plasma to provide PK analysis. 6) Evan's blue dye is quantified by reading absorbency of 200 ul of extracted solution in microtiter plates at 620 nm.
  • Each animal has one IgE anti-DNP sensitized ear (right ear by convention) and one PBS control ear (left ear by convention).
  • Groups 1-8 represent the vehicle and compound testing arms;
  • Group 9 represents the non-antigen negative control;
  • Group 10 represents the non-sensitized challenged negative control;
  • Group 11 represents the non-antigen challenged, non-sensitized negative control group (Groups 9-11 represent negative controls for background levels only and require only minimal number of animals per group.)
  • Example 4 The compounds disclosed in the examples above were tested in the Syk biochemical assay described herein (Example 4) and certain of those compounds exhibited an IC 50 value less than or equal to 1 micromolar. Certain of those compounds exhibited an IC 50 value less than or equal to 100 nM. Certain of those compounds exhibited an IC 50 value less than or equal to 10 nM.
  • Example 3 Some of the compounds disclosed in synthetic Example 3 were tested in the B-cell proliferation assay (as described in Example 6) and exhibited an IC 50 value less than or equal to 10 micromolar. Certain of those compounds exhibited an IC 50 value less than or equal to 1 micromolar.
  • Certain of those compounds did not inhibit T-cell proliferation and had IC 50 values greater than or equal to 5 micromolar when assayed under conditions described herein (as described in Example 7).
  • Certain compounds described herein exhibited IC 50 values for inhibition of T-cell proliferation that were at least 3-fold, and in some instances 5-fold, greater than the IC 50 values of those compounds for inhibition of B-cell proliferation.
  • Some of the compounds described herein were tested in an assay for inhibition of B cell activity (under the conditions described in example 8), and exhibited an IC 50 value less than or equal to 10 micromolar. Certain of those compounds exhibited an IC 50 value less than or equal to 1 micromolar.
  • Some of the compounds disclosed in described herein exhibited both biochemical and cell-based activity. For example, some of the compounds described herein exhibited an IC 50 value less than or equal to 10 micromolar in the Syk biochemical assay described herein (Example 4) and an IC 50 value less than or equal to 10 micromolar in at least one of the cell-based assays (other than the T-cell assay) described herein (Examples 5, 6, 8 or 9).
  • Certain of those compounds exhibited an IC 50 value less than or equal to 1 micromolar in the Syk biochemical assay described herein (Example 4) and an IC 50 value less than or equal to 10 micromolar in at least one of the cell-based assays (other than the T-cell assay) described herein (Examples 5, 6, 8 or 9). Certain of those compounds exhibited an IC 50 value less than or equal to 0.1 micromolar and an IC 50 value less than or equal to 10 micromolar in at least one of the cell-based assays (other than the T-cell assay) described herein (Examples 5, 6, 8 or 9).

Abstract

Imidazopyrazines of Formula I
Figure US08697699-20140415-C00001
and pharmaceutically acceptable salts and mixtures thereof, are provided herein. Pharmaceutical compositions comprising at least one chemical entity and one or more pharmaceutically acceptable vehicle are also provided. Methods of treating patients suffering from certain diseases and disorders responsive to the inhibition of Syk activity, which comprises administering to such patients an amount of at least one chemical entity effective to reduce signs or symptoms of the disease or disorder are disclosed. These diseases include cancer (e.g., B-cell lymphoma and leukemia), autoimmune diseases, inflammatory diseases, acute inflammatory reactions, and allergic disorders. Methods of treatment include administering at least one chemical entity as a single active agent or administering at least one chemical entity in combination with one or more other therapeutic agents. Also provided are methods for determining the presence or absence of Syk kinase in a sample.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims priority to U.S. patent application Ser. No. 12/370,103, filed Feb. 12, 2009, which claims priority to U.S. Provisional Application No. 61/028,463, filed Feb. 13, 2008, all of which are incorporated herein by reference for all purposes.
FIELD
Provided herein are certain substituted amides, compositions, and methods of their use.
BACKGROUND
Protein kinases, the largest family of human enzymes, encompass well over 500 proteins. Spleen Tyrosine Kinase (Syk) is a member of the Syk family of tyrosine kinases, and is a regulator of early B-cell development as well as mature B-cell activation, signaling, and survival.
Syk is a non-receptor tyrosine kinase that plays critical roles in immunoreceptor- and integrin-mediated signaling in a variety of cell types, including B cells, macrophages, monocytes, mast cells, eosinophils, basophils, neutrophils, dendritic cells, T cells, natural killer cells, platelets, and osteoclasts. Immunoreceptors as described here include classical immunoreceptors and immunoreceptor-like molecules. Classical immunoreceptors include B-cell and T-cell antigen receptors as well as various immunoglobulin receptors (Fc receptors). Immunoreceptor-like molecules are either structurally related to immunoreceptors or participate in similar signal transduction pathways and are primarily involved in non-adaptive immune functions, including neutrophil activation, natural killer cell recognition, and osteoclast activity. Integrins are cell surface receptors that play key roles in the control of leukocyte adhesion and activation in both innate and adaptive immunity.
Ligand binding leads to activation of both immunoreceptors and integrins, which results in Src family kinases being activated, and phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs) in the cytoplasmic face of receptor-associated transmembrane adaptors. Syk binds to the phosphorylated ITAM motifs of the adaptors, leading to activation of Syk and subsequent phosphorylation and activation of downstream signaling pathways.
Syk is essential for B-cell activation through B-cell receptor (BCR) signaling. SYK becomes activated upon binding to phosphoryated BCR and thus initiates the early signaling events following BCR activation. B-cell signaling through BCR can lead to a wide range of biological outputs, which in turn depend on the developmental stage of the B-cell. The magnitude and duration of BCR signals must be precisely regulated. Aberrant BCR-mediated signaling can cause disregulated B-cell activation and/or the formation of pathogenic auto-antibodies leading to multiple autoimmune and/or inflammatory diseases. Mice lacking Syk show impaired maturation of B-cells, diminished immunoglobulin production, compromised T-cell-independent immune responses and marked attenuation of the sustained calcium sign upon BCR stimulation.
A large body of evidence supports the role of B-cells and the humoral immune system in the pathogenesis of autoimmune and/or inflammatory diseases. Protein-based therapeutics (such as Rituxan) developed to deplete B-cells represent an approach to the treatment of a number of autoimmune and inflammatory diseases. Auto-antibodies and their resulting immune complexes are known to play pathogenic roles in autoimmune disease and/or inflammatory disease. The pathogenic response to these antibodies is dependent on signaling through Fc Receptors, which is, in turn, dependent upon Syk. Because of Syk's role in B-cell activation, as well as FcR dependent signaling, inhibitors of Syk can be useful as inhibitors of B-cell mediated pathogenic activity, including autoantibody production. Therefore, inhibition of Syk enzymatic activity in cells is proposed as a treatment for autoimmune disease through its effects on autoantibody production.
Syk also plays a key role in FcεRI mediated mast cell and eosinophil activation. Thus, Syk is implicated in allergic disorders including asthma. Syk binds to ITAM motifs on the phosphorylated gamma chain of FcεRI via its SH2 domains and is essential for downstream signaling. Syk deficiency in mice is associated with impaired IgE-mediated mast cell activation as marked by defective degranulation, leukotriene release, and cytokine secretion (e.g. TNF-alpha). Pharmacologic agents that inhibit Syk activity have also been shown to inhibit mast cell degranulation, leukotriene release, and cytokine secretion in cell based assays. Additionally, Syk inhibitors have been shown to inhibit antigen-induced passive cutaneous anaphylaxsis, bronchoconstriction and bronchial edema in rats. Treatment with Syk antisense oligonucleotides inhibits antigen-induced infiltration of eosinophils and neutrophils in an animal model of asthma. Syk deficient eosinophils also show impaired activation in response to FcεRI stimulation. Therefore, small molecule inhibitors of Syk will be useful for treatment of allergy-induced inflammatory diseases including asthma.
Thus, the inhibition of Syk activity can be useful for the treatment of allergic disorders, autoimmune diseases and inflammatory diseases such as: SLE, rheumatoid arthritis, multiple vasculitides, idiopathic thrombocytopenic purpura (ITP), myasthenia gravis, allergic rhinitis, chronic obstructive pulmonary disease (COPD), adult respiratory distress syndrome (ARDs) and asthma. In addition, Syk has been reported to play an important role in ligand-independent tonic signaling through the B-cell receptor, known to be an important survival signal in B-cells. Thus, inhibition of Syk activity may be useful in treating certain types of cancer, including B-cell lymphoma and leukemia.
BRIEF SUMMARY
Provided is at least one chemical entity chosen from compounds of Formula I:
Figure US08697699-20140415-C00002
    • and pharmaceutically acceptable salts thereof, wherein:
    • W is selected from optionally substituted aryl and optionally substituted heteroaryl;
    • A is selected from optionally substituted alkyl, optionally substituted alkoxy, optionally substituted amino, carboxy, cyan, halo, hydrogen, hydroxy and nitro;
    • X is selected from CH and N;
    • Q is selected from CO and SO2;
    • Y is selected from hydrogen, optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, and optionally substituted heterocycloalkyl;
    • Z is selected from hydrogen, optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, and optionally substituted heterocycloalkyl; or
    • Z taken together with Y, and the nitrogen to which they are bound, form an optionally substituted 5- to 13-membered nitrogen-containing ring which optionally includes one or two additional heteroatoms chosen from O and S;
    • provided that if W is 4-(morpholino)phenyl, A is hydrogen, X is CH, Q is CO and Y is hydrogen, then Z is not hydroxyethyl;
    • provided that if W is p-chlorophenyl or 4-(p-tolylsulfonamido)phenyl, A is hydrogen, X is CH, Q is CO and Y is hydrogen, then Z is not N,N-dimethylaminoethyl; and
    • provided that if W is 4-(morpholinecarbonyl)phenyl, A is hydrogen, X is CH, Q is CO and Y is hydrogen, then Z is not 3-chlorobenzyl or 4-t-butylphenyl.
Provided is at least one chemical entity chosen from compounds of Formula I
Figure US08697699-20140415-C00003
    • and pharmaceutically acceptable salts thereof, wherein:
    • W is selected from optionally substituted aryl and optionally substituted heteroaryl;
    • A is selected from optionally substituted alkyl, optionally substituted alkoxy, optionally substituted amino, carboxy, cyano, halo, hydrogen, hydroxy and nitro;
    • X is selected from CH and N;
    • Q is selected from CO and SO2;
    • Y is selected from hydrogen, optionally substituted alkyl; optionally substituted cycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, and optionally substituted heterocycloalkyl;
    • Z is selected from hydrogen, optionally substituted alkyl; optionally substituted cycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, and optionally substituted heterocycloalkyl; or
    • Z taken together with Y, and the nitrogen to which they are bound, form a 5- to 13-membered nitrogen-containing ring;
    • provided that if W is 4-(morpholino)phenyl, A is hydrogen, X is CH, Q is CO and Y is hydrogen, then Z is not hydroxyethyl;
    • provided that if W is p-chlorophenyl or 4-(p-tolylsulfonamido)phenyl, A is hydrogen, X is CH, Q is CO and Y is hydrogen, then Z is not N,N-dimethylaminoethyl; and
    • provided that if W is 4-(morpholinecarbonyl)phenyl, A is hydrogen, X is CH, Q is CO and Y is hydrogen, then Z is not 3-chlorobenzyl or 4-t-butylphenyl.
Also provided is a pharmaceutical composition, comprising at least one chemical entity described herein, together with at least one pharmaceutically acceptable vehicle chosen from carriers, adjuvants, and excipients.
Also provided is a method for treating a patient having a disease responsive to inhibition of Syk activity, comprising administering to the patient an effective amount of at least one chemical entity described herein.
Also provided is a method for treating a patient having a disease chosen from cancer, autoimmune diseases, inflammatory diseases, acute inflammatory reactions, and allergic disorders comprising administering to the patient an effective amount of at least one chemical entity described herein.
Also provided is a method for increasing sensitivity of cancer cells to chemotherapy, comprising administering to a patient undergoing chemotherapy with a chemotherapeutic agent an amount of at least one chemical entity described herein, sufficient to increase the sensitivity of cancer cells to the chemotherapeutic agent.
Also provided is a method for inhibiting ATP hydrolysis, the method comprising contacting cells expressing Syk with at least one chemical entity described herein in an amount sufficient to detectably decrease the level of ATP hydrolysis in vitro.
Also provided is a method for determining the presence of Syk in a sample, comprising contacting the sample with at least one chemical entity described herein under conditions that permit detection of Syk activity, detecting a level of Syk activity in the sample, and therefrom determining the presence or absence of Syk in the sample.
Also provided is a method for inhibiting B-cell activity comprising contacting cells expressing Syk with at least one chemical entity described herein in an amount sufficient to detectably decrease B-cell activity in vitro.
DETAILED DESCRIPTION
As used herein, when any variable occurs more than one time in a chemical formula, its definition on each occurrence is independent of its definition at every other occurrence. In accordance with the usual meaning of “a” and “the” in patents, reference, for example, to “a” kinase or “the” kinase is inclusive of one or more kinases.
As used in the present specification, the following words, phrases and symbols are generally intended to have the meanings as set forth below, except to the extent that the context in which they are used indicates otherwise. The following abbreviations and terms have the indicated meanings throughout:
A dash (“-”) that is not between two letters or symbols is used to indicate a point of attachment for a substituent. For example, —CONH2 is attached through the carbon atom.
By “optional” or “optionally” is meant that the subsequently described event or circumstance may or may not occur, and that the description includes instances where the event or circumstance occurs and instances in which it does not. For example, “optionally substituted alkyl” encompasses both “alkyl” and “substituted alkyl” as defined below. It will be understood by those skilled in the art, with respect to any group containing one or more substituents, that such groups are not intended to introduce any substitution or substitution patterns that are sterically impractical, synthetically non-feasible and/or inherently unstable.
“Alkyl” encompasses straight chain and branched chain having the indicated number of carbon atoms, usually from 1 to 20 carbon atoms, for example 1 to 8 carbon atoms, such as 1 to 6 carbon atoms. For example C1-C6 alkyl encompasses both straight and branched chain alkyl of from 1 to 6 carbon atoms. Examples of alkyl groups include methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, pentyl, 2-pentyl, isopentyl, neopentyl, hexyl, 2-hexyl, 3-hexyl, 3-methylpentyl, and the like. Alkylene is another subset of alkyl, referring to the same residues as alkyl, but having two points of attachment. Alkylene groups will usually have from 2 to 20 carbon atoms, for example 2 to 8 carbon atoms, such as from 2 to 6 carbon atoms. For example, C0 alkylene indicates a covalent bond and C1 alkylene is a methylene group. When an alkyl residue having a specific number of carbons is named, all geometric isomers having that number of carbons are intended to be encompassed; thus, for example, “butyl” is meant to include n-butyl, sec-butyl, isobutyl and t-butyl; “propyl” includes n-propyl and isopropyl. “Lower alkyl” refers to alkyl groups having 1 to 4 carbons.
“Alkenyl” indicates an unsaturated branched or straight-chain alkyl group having at least one carbon-carbon double bond derived by the removal of one molecule of hydrogen from adjacent carbon atoms of the parent alkyl. The group may be in either the cis or trans configuration about the double bond(s). Typical alkenyl groups include, but are not limited to, ethenyl; propenyls such as prop-1-en-1-yl, prop-1-en-2-yl, prop-2-en-1-yl (allyl), prop-2-en-2-yl; butenyls such as but-1-en-1-yl, but-1-en-2-yl, 2-methyl-prop-1-en-1-yl, but-2-en-1-yl, but-2-en-1-yl, but-2-en-2-yl, buta-1,3-dien-1-yl, buta-1,3-dien-2-yl; and the like. In some embodiments, an alkenyl group has from 2 to 20 carbon atoms and in other embodiments, from 2 to 6 carbon atoms.
“Alkynyl” indicates an unsaturated branched or straight-chain alkyl group having at least one carbon-carbon triple bond derived by the removal of two molecules of hydrogen from adjacent carbon atoms of the parent alkyl. Typical alkynyl groups include, but are not limited to, ethynyl; propynyls such as prop-1-yn-1-yl, prop-2-yn-1-yl; butynyls such as but-1-yn-1-yl, but-1-yn-3-yl, but-3-yn-1-yl; and the like. In some embodiments, an alkynyl group has from 2 to 20 carbon atoms and in other embodiments, from 3 to 6 carbon atoms.
The term “carboxy” refers to the group —C(═O)OH.
“Cycloalkyl” indicates a saturated hydrocarbon ring group, having the specified number of carbon atoms, usually from 3 to 7 ring carbon atoms. Examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl as well as bridged and caged saturated ring groups such as norbornane.
The term “cycloalkyloxy” refers to the group —O-cycloalkyl.
By “alkoxy” is meant an alkyl group of the indicated number of carbon atoms attached through an oxygen bridge such as, for example, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, sec-butoxy, tert-butoxy, pentoxy, 2-pentyloxy, isopentoxy, neopentoxy, hexoxy, 2-hexoxy, 3-hexoxy, 3-methylpentoxy, and the like. Alkoxy groups will usually have from 1 to 6 carbon atoms attached through the oxygen bridge. “Lower alkoxy” refers to alkoxy groups having 1 to 4 carbons.
“Aminocarbonyl” encompasses a group of the formula —(C═O)NRaRb where Ra and Rb are independently chosen from hydrogen and the optional substituents for “substituted amino” described below.
“Acyl” refers to the groups (alkyl)-C(O)—; (cycloalkyl)-C(O)—; (aryl)-C(O)—; (heteroaryl)-C(O)—; and (heterocycloalkyl)-C(O)—, wherein the group is attached to the parent structure through the carbonyl functionality and wherein alkyl, cycloalkyl, aryl, heteroaryl, and heterocycloalkyl are as described herein. Acyl groups have the indicated number of carbon atoms, with the carbon of the keto group being included in the numbered carbon atoms. For example a C2 acyl group is an acetyl group having the formula CH3(C═O)—.
By “alkoxycarbonyl” is meant an ester group of the formula (alkoxy)(C═O)— attached through the carbonyl carbon wherein the alkoxy group has the indicated number of carbon atoms. Thus a C1-C6alkoxycarbonyl group is an alkoxy group having from 1 to 6 carbon atoms attached through its oxygen to a carbonyl linker.
By “amino” is meant the group —NH2.
“Aryl” encompasses:
    • 5- and 6-membered carbocyclic aromatic rings, for example, benzene;
    • bicyclic ring systems wherein at least one ring is carbocyclic and aromatic, for example, naphthalene, indane, and tetralin; and
    • tricyclic ring systems wherein at least one ring is carbocyclic and aromatic, for example, fluorene.
      For example, aryl includes 5- and 6-membered carbocyclic aromatic rings fused to a 5- to 7-membered heterocycloalkyl ring containing 1 or more heteroatoms chosen from N, O, and S. For such fused, bicyclic ring systems wherein only one of the rings is a carbocyclic aromatic ring, the point of attachment may be at the carbocyclic aromatic ring or the heterocycloalkyl ring. Bivalent radicals formed from substituted benzene derivatives and having the free valences at ring atoms are named as substituted phenylene radicals. Bivalent radicals derived from univalent polycyclic hydrocarbon radicals whose names end in “-yl” by removal of one hydrogen atom from the carbon atom with the free valence are named by adding “-idene” to the name of the corresponding univalent radical, e.g., a naphthyl group with two points of attachment is termed naphthylidene. Aryl, however, does not encompass or overlap in any way with heteroaryl, separately defined below. Hence, if one or more carbocyclic aromatic rings is fused with a heterocycloalkyl aromatic ring, the resulting ring system is heteroaryl, not aryl, as defined herein.
The term “aryloxy” refers to the group —O-aryl.
The term “halo” includes fluoro, chloro, bromo, and iodo, and the term “halogen” includes fluorine, chlorine, bromine, and iodine.
“Heteroaryl” encompasses:
    • 5- to 7-membered aromatic, monocyclic rings containing one or more, for example, from 1 to 4, or In some embodiments, from 1 to 3, heteroatoms chosen from N, O, and S, with the remaining ring atoms being carbon; and
    • bicyclic heterocycloalkyl rings containing one or more, for example, from 1 to 4, or In some embodiments, from 1 to 3, heteroatoms chosen from N, O, and S, with the remaining ring atoms being carbon and wherein at least one heteroatom is present in an aromatic ring.
      For example, heteroaryl includes a 5- to 7-membered heterocycloalkyl, aromatic ring fused to a 5- to 7-membered cycloalkyl ring. For such fused, bicyclic heteroaryl ring systems wherein only one of the rings contains one or more heteroatoms, the point of attachment may be at the heteroaromatic ring or the cycloalkyl ring. When the total number of S and O atoms in the heteroaryl group exceeds 1, those heteroatoms are not adjacent to one another. In some embodiments, the total number of S and O atoms in the heteroaryl group is not more than 2. In some embodiments, the total number of S and O atoms in the aromatic heterocycle is not more than 1. Examples of heteroaryl groups include, but are not limited to, (as numbered from the linkage position assigned priority 1), 2-pyridyl, 3-pyridyl, 4-pyridyl, 2,3-pyrazinyl, 3,4-pyrazinyl, 2,4-pyrimidinyl, 3,5-pyrimidinyl, 2,3-pyrazolinyl, 2,4-imidazolinyl, isoxazolinyl, oxazolinyl, thiazolinyl, thiadiazolinyl, tetrazolyl, thienyl, benzothiophenyl, furanyl, benzofuranyl, benzoimidazolinyl, indolinyl, pyridizinyl, triazolyl, quinolinyl, pyrazolyl, and 5,6,7,8-tetrahydroisoquinoline. Bivalent radicals derived from univalent heteroaryl radicals whose names end in “-yl” by removal of one hydrogen atom from the atom with the free valence are named by adding “-idene” to the name of the corresponding univalent radical, e.g., a pyridyl group with two points of attachment is a pyridylidene. Heteroaryl does not encompass or overlap with aryl as defined above.
Substituted heteroaryl also includes ring systems substituted with one or more oxide (—O−) substituents, such as pyridinyl N-oxides.
The term “heteroaryloxy” refers to the group —O-heteroaryl.
By “heterocycloalkyl” is meant a single aliphatic ring, usually with 3 to 7 ring atoms, containing at least 2 carbon atoms in addition to 1-3 heteroatoms independently selected from oxygen, sulfur, and nitrogen, as well as combinations comprising at least one of the foregoing heteroatoms. Suitable heterocycloalkyl groups include, for example (as numbered from the linkage position assigned priority 1), 2-pyrrolinyl, 2,4-imidazolidinyl, 2,3-pyrazolidinyl, 2-piperidyl, 3-piperidyl, 4-piperdyl, and 2,5-piperzinyl. Morpholinyl groups are also contemplated, including 2-morpholinyl and 3-morpholinyl (numbered wherein the oxygen is assigned priority 1). Substituted heterocycloalkyl also includes ring systems substituted with one or more oxo moieties, such as piperidinyl N-oxide, morpholinyl-N-oxide, 1-oxo-1-thiomorpholinyl and 1,1-dioxo-1-thiomorpholinyl.
The term “heterocycloalkyloxy” refers to the group —O-heterocylcoalkyl.
The term “nitro” refers to the group —NO2.
The term “phosphono” refers to the group —PO3H2.
“Thiocarbonyl” refers to the group —C(═O)SH.
The term “optionally substituted thiocarbonyl” includes the following groups: —C(═O)S-(optionally substituted (C1-C6)alkyl), —C(═O)S-(optionally substituted aryl), —C(═O)S-(optionally substituted heteroaryl), and —C(═O)S-(optionally substituted heterocycloalkyl).
The term “sulfanyl” includes the groups: —S-(optionally substituted (C1-C6)alkyl), —S-(optionally substituted aryl), —S-(optionally substituted heteroaryl), and —S-(optionally substituted heterocycloalkyl). Hence, sulfanyl includes the group C1-C6 alkylsulfanyl.
The term “sulfinyl” includes the groups: —S(O)—H, —S(O)-(optionally substituted (C1-C6)alkyl), —S(O)-optionally substituted aryl), —S(O)-optionally substituted heteroaryl), —S(O)-(optionally substituted heterocycloalkyl); and —S(O)-(optionally substituted amino).
The term “sulfonyl” includes the groups: —S(O2)—H, —S(O2)-(optionally substituted (C1-C6)alkyl), —S(O2)-optionally substituted aryl), —S(O2)-optionally substituted heteroaryl), —S(O2)-(optionally substituted heterocycloalkyl), —S(O2)-(optionally substituted alkoxy), —S(O2)-optionally substituted aryloxy), —S(O2)-optionally substituted heteroaryloxy), —S(O2)-(optionally substituted heterocyclyloxy); and —S(O2)-(optionally substituted amino).
The term “substituted”, as used herein, means that any one or more hydrogens on the designated atom or group is replaced with a selection from the indicated group, provided that the designated atom's normal valence is not exceeded. When a substituent is oxo (i.e., ═O) then 2 hydrogens on the atom are replaced. Combinations of substituents and/or variables are permissible only if such combinations result in stable compounds or useful synthetic intermediates. A stable compound or stable structure is meant to imply a compound that is sufficiently robust to survive isolation from a reaction mixture, and subsequent formulation as an agent having at least practical utility. Unless otherwise specified, substituents are named into the core structure. For example, it is to be understood that when (cycloalkyl)alkyl is listed as a possible substituent, the point of attachment of this substituent to the core structure is in the alkyl portion.
The terms “substituted” alkyl, cycloalkyl, aryl, heterocycloalkyl, and heteroaryl, unless otherwise expressly defined, refer respectively to alkyl, cycloalkyl, aryl, heterocycloalkyl, and heteroaryl wherein one or more (such as up to 5, for example, up to 3) hydrogen atoms are replaced by a substituent independently chosen from:
    • —Ra, —ORb, —O(C1-C2 alkyl)O— (e.g., methylenedioxy-), —SRb, guanidine, guanidine wherein one or more of the guanidine hydrogens are replaced with a lower-alkyl group, —NRbRc, halo, cyano, nitro, —CORb, —CO2Rb, —CONRbRc, OCORb, —OCO2Ra, —OCONRbRc, —NRcCORb, —NRcCO2Ra, —NRcCONRbRc, —CO2Rb, —CONRbRc, —NRcCORb, —SORa, —SO2Ra, —SO2NRbRc, and —NRcSO2Ra,
    • where Ra is chosen from optionally substituted C1-C6 alkyl, optionally substituted aryl, and optionally substituted heteroaryl;
    • Rb is chosen from H, optionally substituted C1-C6 alkyl, optionally substituted aryl, and optionally substituted heteroaryl; and
    • Rc is chosen from hydrogen and optionally substituted C1-C4 alkyl; or
    • Rb and Rc, and the nitrogen to which they are attached, form an optionally substituted heterocycloalkyl group; and
    • where each optionally substituted group is unsubstituted or independently substituted with one or more, such as one, two, or three, substituents independently selected from C1-C4 alkyl, aryl, heteroaryl, aryl-C1-C4 alkyl-, heteroaryl-C1-C4 alkyl-, C1-C4 haloalkyl-, —OC1-C4 alkyl, —OC1-C4 alkylphenyl, —C1-C4 alkyl-OH, —OC1-C4 haloalkyl, halo, —OH, —NH2, —C1-C4 alkyl-NH2, —N(C1-C4 alkyl)(C1-C4 alkyl), —NH(C1-C4 alkyl), —N(C1-C4 alkyl)(C1-C4 alkylphenyl), —NH(C1-C4 alkylphenyl), cyano, nitro, oxo (as a substitutent for heteroaryl), —CO2H, —C(O)OC1-C4 alkyl, —CON(C1-C4 alkyl)(C1-C4 alkyl), —CONH(C1-C4 alkyl), —CONH2, —NHC(O)(C1-C4 alkyl), —NHC(O)(phenyl), —N(C1-C4 alkyl)C(O)(C1-C4 alkyl), —N(C1-C4 alkyl)C(O)(phenyl), —C(O)C1-C4 alkyl, —C(O)C1-C4 phenyl, —C(O)C1-C4 haloalkyl, —OC(O)C1-C4 alkyl, —SO2(C1-C4 alkyl), —SO2(phenyl), —SO2(C1-C4 haloalkyl), —SO2NH2, —SO2NH(C1-C4 alkyl), —SO2NH(phenyl), —NHSO2(C1-C4 alkyl), —NHSO2(phenyl), and —NHSO2(C1-C4 haloalkyl).
The term “substituted acyl” refers to the groups (substituted alkyl)-C(O)—; (substituted cycloalkyl)-C(O)—; (substituted aryl)-C(O)—; (substituted heteroaryl)-C(O)—; and (substituted heterocycloalkyl)-C(O)—, wherein the group is attached to the parent structure through the carbonyl functionality and wherein substituted alkyl, cycloalkyl, aryl, heteroaryl, and heterocycloalkyl, refer respectively to alkyl, cycloalkyl, aryl, heteroaryl, and heterocycloalkyl wherein one or more (such as up to 5, for example, up to 3) hydrogen atoms are replaced by a substituent independently chosen from:
    • Ra, —ORb, —O(C1-C2 alkyl)O— (e.g., methylenedioxy-), —SRb, guanidine, guanidine wherein one or more of the guanidine hydrogens are replaced with a lower-alkyl group, —NRbRc, halo, cyano, nitro, —CORb, —CO2Rb, —CONRbRc, —OCORb, —OCO2Ra, —OCONRbRc, —NRcCORb, —NRcCO2Ra, —NRcCONRbRc, —CO2Rb, —CONRbRc, —NRcCORb, —SORa, —SO2Ra, —SO2NRbRc, and —NRcSO2Ra,
    • where Ra is chosen from optionally substituted C1-C6 alkyl, optionally substituted aryl, and optionally substituted heteroaryl;
    • Rb is chosen from H, optionally substituted C1-C6 alkyl, optionally substituted aryl, and optionally substituted heteroaryl; and
    • Rc is chosen from hydrogen and optionally substituted C1-C4 alkyl; or
    • Rb and Rc, and the nitrogen to which they are attached, form an optionally substituted heterocycloalkyl group; and
    • where each optionally substituted group is unsubstituted or independently substituted with one or more, such as one, two, or three, substituents independently selected from C1-C4 alkyl, aryl, heteroaryl, aryl-C1-C4 alkyl-, heteroaryl-C1-C4 alkyl-, C1-C4 haloalkyl-, —OC1-C4 alkyl, —OC1-C4 alkylphenyl, —C1-C4 alkyl-OH, —OC1-C4 haloalkyl, halo, —OH, —NH2, —C1-C4 alkyl-NH2, —N(C1-C4 alkyl)(C1-C4 alkyl), —NH(C1-C4 alkyl), —N(C1-C4 alkyl)(C1-C4 alkylphenyl), —NH(C1-C4 alkylphenyl), cyano, nitro, oxo (as a substitutent for heteroaryl), —CO2H, —C(O)OC1-C4 alkyl, —CON(C1-C4 alkyl)(C1-C4 alkyl), —CONH(C1-C4 alkyl), —CONH2, —NHC(O)(C1-C4 alkyl), —NHC(O)(phenyl), —N(C1-C4 alkyl)C(O)(C1-C4 alkyl), —N(C1-C4 alkyl)C(O)(phenyl), —C(O)C1-C4 alkyl, —C(O)C1-C4 phenyl, —C(O)C1-C4 haloalkyl, —OC(O)C1-C4 alkyl, —SO2(C1-C4 alkyl), —SO2(phenyl), —SO2(C1-C4haloalkyl), —SO2NH2, —SO2NH(C1-C4 alkyl), —SO2NH(phenyl), —NHSO2(C1-C4 alkyl), —NHSO2(phenyl), and —NHSO2(C1-C4haloalkyl).
The term “substituted alkoxy” refers to alkoxy wherein the alkyl constituent is substituted (i.e., —O-(substituted alkyl)) wherein “substituted alkyl” refers to alkyl wherein one or more (such as up to 5, for example, up to 3) hydrogen atoms are replaced by a substituent independently chosen from:
    • —Ra, —ORb, —O(C1-C2 alkyl)O— (e.g., methylenedioxy-), —SRb, guanidine, guanidine wherein one or more of the guanidine hydrogens are replaced with a lower-alkyl group, —NRbRc, halo, cyano, nitro, —CORb, —CO2Rb, —CONRbRc, —OCORb, —OCO2Ra, —OCONRbRc, —NRcCORb, —NRcCO2Ra, —NRcCONRbRc, —CO2Rb, —CONRbRc, —NRcCORb, —SORa, —SO2Ra, —SO2NRbRc, and —NRcSO2Ra,
    • where Ra is chosen from optionally substituted C1-C6 alkyl, optionally substituted aryl, and optionally substituted heteroaryl;
    • Rb is chosen from H, optionally substituted C1-C6 alkyl, optionally substituted aryl, and optionally substituted heteroaryl; and
    • Rc is chosen from hydrogen and optionally substituted C1-C4 alkyl; or
    • Rb and Rc, and the nitrogen to which they are attached, form an optionally substituted heterocycloalkyl group; and
    • where each optionally substituted group is unsubstituted or independently substituted with one or more, such as one, two, or three, substituents independently selected from C1-C4 alkyl, aryl, heteroaryl, aryl-C1-C4 alkyl-, heteroaryl-C1-C4 alkyl-, C1-C4 haloalkyl-, —OC1-C4 alkyl, —OC1-C4 alkylphenyl, —C1-C4 alkyl-OH, haloalkyl, halo, —OH, —NH2, —C1-C4 alkyl-NH2, —N(C1-C4alkyl)(C1-C4 alkyl), —NH(C1-C4 alkyl), —N(C1-C4 alkyl)(C1-C4 alkylphenyl), —NH(C1-C4 alkylphenyl), cyano, nitro, oxo (as a substitutent for heteroaryl), —CO2H, —C(O)OC1-C4 alkyl, —CON(C1-C4 alkyl)(C1-C4 alkyl), —CONH(C1-C4 alkyl), —CONH2, —NHC(O)(C1-C4 alkyl), —NHC(O)(phenyl), —N(C1-C4 alkyl)C(O)(C1-C4 alkyl), —N(C1-C4 alkyl)C(O)(phenyl), —C(O)C1-C4 alkyl, —C(O)C1-C4 phenyl, —C(O)C1-C4 haloalkyl, —OC(O)C1-C4 alkyl, —SO2(C1-C4 alkyl), —SO2(phenyl), —SO2(C1-C4 haloalkyl), —SO2NH2, —SO2NH(C1-C4 alkyl), —SO2NH(phenyl), —NHSO2(C1-C4 alkyl), —NHSO2(phenyl), and —NHSO2(C1-C4 haloalkyl). In some embodiments, a substituted alkoxy group is “polyalkoxy” or —O-(optionally substituted alkylene)-(optionally substituted alkoxy), and includes groups such as —OCH2CH2OCH3, and residues of glycol ethers such as polyethyleneglycol, and —O(CH2CH2O)xCH3, where x is an integer of 2-20, such as 2-10, and for example, 2-5. Another substituted alkoxy group is hydroxyalkoxy or —OCH2(CH2)yOH, where y is an integer of 1-10, such as 1-4.
The term “substituted alkoxycarbonyl” refers to the group (substituted alkyl)-O—C(O)— wherein the group is attached to the parent structure through the carbonyl functionality and wherein substituted refers to alkyl wherein one or more (such as up to 5, for example, up to 3) hydrogen atoms are replaced by a substituent independently chosen from:
    • —Ra, —ORb, —O(C1-C2 alkyl)O— (e.g., methylenedioxy-), —SRb, guanidine, guanidine wherein one or more of the guanidine hydrogens are replaced with a lower-alkyl group, —NRbRc, halo, cyano, nitro, —CORb, —CO2Rb, —CONRbRc, —OCORb, —OCO2Ra, —OCONRbRc, —NRcCORb, —NRcCO2Ra, —NRcCONRbRc, —CO2Rb, —CONRbRc, —NRcCORb, —SORa, —SO2Ra, —SO2NRbRc, and —NRcSO2Ra,
    • where Ra is chosen from optionally substituted C1-C6 alkyl, optionally substituted aryl, and optionally substituted heteroaryl;
    • Rb is chosen from H, optionally substituted C1-C6 alkyl, optionally substituted aryl, and optionally substituted heteroaryl; and
    • Rc is chosen from hydrogen and optionally substituted C1-C4 alkyl; or
    • Rb and Rc, and the nitrogen to which they are attached, form an optionally substituted heterocycloalkyl group; and
    • where each optionally substituted group is unsubstituted or independently substituted with one or more, such as one, two, or three, substituents independently selected from C1-C4 alkyl, aryl, heteroaryl, aryl-C1-C4 alkyl-, heteroaryl-C1-C4 alkyl-, C1-C4 haloalkyl-, —OC1-C4 alkyl, —OC1-C4 alkylphenyl, —C1-C4 alkyl-OH, —OC1-C4 haloalkyl, halo, —OH, —NH2, —C1-C4 alkyl-NH2, —N(C1-C4 alkyl)(C1-C4 alkyl), —NH(C1-C4 alkyl), —N(C1-C4 alkyl)(C1-C4 alkylphenyl), —NH(C1-C4 alkylphenyl), cyano, nitro, oxo (as a substitutent for heteroaryl), —CO2H, —C(O)OC1-C4 alkyl, —CON(C1-C4 alkyl)(C1-C4 alkyl), —CONH(C1-C4 alkyl), —CONH2, —NHC(O)(C1-C4 alkyl), —NHC(O)(phenyl), —N(C1-C4 alkyl)C(O)(C1-C4 alkyl), —N(C1-C4 alkyl)C(O)(phenyl), —C(O)C1-C4 alkyl, —C(O)C1-C4 phenyl, —C(O)C1-C4 haloalkyl, —OC(O)C1-C4 alkyl, —SO2(C1-C4 alkyl), —SO2(phenyl), —SO2(C1-C4 haloalkyl), —SO2NH2, —SO2NH(C1-C4 alkyl), —SO2NH(phenyl), —NHSO2(C1-C4 alkyl), —NHSO2(phenyl), and —NHSO2(C1-C4 haloalkyl).
The term “substituted aryloxy” refers to aryloxy wherein the aryl constituent is substituted (i.e., —O-(substituted aryl)) wherein “substituted aryl” refers to aryl wherein one or more (such as up to 5, for example, up to 3) hydrogen atoms are replaced by a substituent independently chosen from:
    • —Ra, —ORb, —O(C1-C2 alkyl)O— (e.g., methylenedioxy-), —SRb, guanidine, guanidine wherein one or more of the guanidine hydrogens are replaced with a lower-alkyl group, —NRbRc, halo, cyano, nitro, —CORb, —CO2Rb, —CONRbRc, —OCORb, —OCO2Ra, —OCONRbRc, —NRcCORb, —NRcCO2Ra, —NRcCONRbRc, —CO2Rb, —CONRbRc, —NRcCORb, —SORa, —SO2Ra, —SO2NRbRc, and —NRcSO2Ra,
    • where Ra is chosen from optionally substituted C1-C6 alkyl, optionally substituted aryl, and optionally substituted heteroaryl;
    • Rb is chosen from H, optionally substituted C1-C6 alkyl, optionally substituted aryl, and optionally substituted heteroaryl; and
    • Rc is chosen from hydrogen and optionally substituted C1-C4 alkyl; or
    • Rb and Rc, and the nitrogen to which they are attached, form an optionally substituted heterocycloalkyl group; and
      where each optionally substituted group is unsubstituted or independently substituted with one or more, such as one, two, or three, substituents independently selected from C1-C4 alkyl, aryl, heteroaryl, aryl-C1-C4 alkyl-, heteroaryl-C1-C4 alkyl-, C1-C4 haloalkyl-, —OC1-C4 alkyl, —OC1-C4 alkylphenyl, —C1-C4 alkyl-OH, —OC1-C4 haloalkyl, halo, —OH, —NH2, —C1-C4 alkyl-NH2, —N(C1-C4 alkyl)(C1-C4 alkyl), —NH(C1-C4 alkyl), —N(C1-C4 alkyl)(C1-C4 alkylphenyl), —NH(C1-C4 alkylphenyl), cyano, nitro, oxo (as a substitutent for heteroaryl), —CO2H, —C(O)OC1-C4 alkyl, —CON(C1-C4 alkyl)(C1-C4 alkyl), —CONH(C1-C4 alkyl), —CONH2, —NHC(O)(C1-C4 alkyl), —NHC(O)(phenyl), —N(C1-C4 alkyl)C(O)(C1-C4 alkyl), —N(C1-C4 alkyl)C(O)(phenyl), —C(O)C1-C4 alkyl, —C(O)C1-C4 phenyl, —C(O)C1-C4 haloalkyl, —OC(O)C1-C4 alkyl, —SO2(C1-C4 alkyl), —SO2(phenyl), —SO2(C1-C4 haloalkyl), —SO2NH2, —SO2NH(C1-C4 alkyl), —SO2NH(phenyl), —NHSO2(C1-C4 alkyl), —NHSO2(phenyl), and —NHSO2(C1-C4 haloalkyl).
The term “substituted heteroaryloxy” refers to heteroaryloxy wherein the aryl constituent is substituted (i.e., —O-(substituted heteroaryl)) wherein “substituted heteroaryl” refers to aryl wherein one or more (such as up to 5, for example, up to 3) hydrogen atoms are replaced by a substituent independently chosen from:
    • Ra, —ORb, —O(C1-C2 alkyl)O— (e.g., methylenedioxy-), —SRb, guanidine, guanidine wherein one or more of the guanidine hydrogens are replaced with a lower-alkyl group, —NRbRc, halo, cyano, nitro, —CORb, —CO2Rb, —CONRbRc, —OCORb, —OCO2Ra, —OCONRbRc, —NRcCORb, —NRcCO2Ra, —NRaCONRbRc, —CO2Rb, —CONRbRa, —NRcCORb, —SORa, —SO2Ra, —SO2NRbRc, and —NRcSO2Ra,
    • where Ra is chosen from optionally substituted C1-C6 alkyl, optionally substituted aryl, and optionally substituted heteroaryl;
    • Rb is chosen from H, optionally substituted C1-C6 alkyl, optionally substituted aryl, and optionally substituted heteroaryl; and
    • Rc is chosen from hydrogen and optionally substituted C1-C4 alkyl; or
    • Rb and Rc, and the nitrogen to which they are attached, form an optionally substituted heterocycloalkyl group; and
      where each optionally substituted group is unsubstituted or independently substituted with one or more, such as one, two, or three, substituents independently selected from C1-C4 alkyl, aryl, heteroaryl, aryl-C1-C4 alkyl-, heteroaryl-C1-C4 alkyl-, C1-C4 haloalkyl-, —OC1-C4 alkyl, —OC1-C4 alkylphenyl, —C1-C4 alkyl-OH, —OC1-C4 haloalkyl, halo, —OH, —NH2, —C1-C4 alkyl-NH2, —N(C1-C4 alkyl)(C1-C4 alkyl), —NH(C1-C4 alkyl), —N(C1-C4 alkyl)(C1-C4 alkylphenyl), —NH(C1-C4 alkylphenyl), cyano, nitro, oxo (as a substitutent for heteroaryl), —CO2H, —C(O)OC1-C4 alkyl, —CON(C1-C4 alkyl)(C1-C4 alkyl), —CONH(C1-C4 alkyl), —CONH2, —NHC(O)(C1-C4 alkyl), —NHC(O)(phenyl), —N(C1-C4 alkyl)C(O)(C1-C4 alkyl), —N(C1-C4 alkyl)C(O)(phenyl), —C(O)C1-C4 alkyl, —C(O)C1-C4 phenyl, —C(O)C1-C4 haloalkyl, —OC(O)C1-C4 alkyl, —SO2(C1-C4 alkyl), —SO2(phenyl), —SO2(C1-C4 haloalkyl), —SO2NH2, —SO2NH(C1-C4 alkyl), —SO2NH(phenyl), —NHSO2(C1-C4 alkyl), —NHSO2(phenyl), and —NHSO2(C1-C4 haloalkyl).
The term “substituted cycloalkyloxy” refers to cycloalkyloxy wherein the cycloalkyl constituent is substituted (i.e., —O-(substituted cycloalkyl)) wherein “substituted cycloalkyl” refers to a cycloalkyl wherein one or more hydrogen atoms are replaced by a substituent independently chosen from:
    • —Ra, —ORb, —O(C1-C2 alkyl)O— (e.g., methylenedioxy-), —SRb, guanidine, guanidine wherein one or more of the guanidine hydrogens are replaced with a lower-alkyl group, —NRbRc, halo, cyano, nitro, —CORb, —CO2Rb, —CONRbRc, —OCORb, —OCO2Ra, —OCONRbRc, —NRcCORb, —NRcCO2Ra, —NRcCONRbRc, —CO2Rb, —CONRbRc, —NRcCORb, —SORa, —SO2Ra, —SO2NRbRc, and —NRcSO2Ra,
    • where Ra is chosen from optionally substituted C1-C6 alkyl, optionally substituted aryl, and optionally substituted heteroaryl;
    • Rb is chosen from H, optionally substituted C1-C6 alkyl, optionally substituted aryl, and optionally substituted heteroaryl; and
    • Rc is chosen from hydrogen and optionally substituted C1-C4 alkyl; or
    • Rb and Rc, and the nitrogen to which they are attached, form an optionally substituted heterocycloalkyl group; and
      where each optionally substituted group is unsubstituted or independently substituted with one or more, such as one, two, or three, substituents independently selected from C1-C4 alkyl, aryl, heteroaryl, aryl-C1-C4 alkyl-, heteroaryl-C1-C4 alkyl-, C1-C4 haloalkyl-, —OC1-C4 alkyl, —OC1-C4 alkylphenyl, —C1-C4 alkyl-OH, haloalkyl, halo, —OH, —NH2, —C1-C4 alkyl-NH2, —N(C1-C4 alkyl)(C1-C4 alkyl), —NH(C1-C4 alkyl), —N(C1-C4 alkyl)(C1-C4 alkylphenyl), —NH(C1-C4 alkylphenyl), cyano, nitro, oxo (as a substitutent for heteroaryl), —CO2H, —C(O)OC1-C4 alkyl, —CON(C1-C4 alkyl)(C1-C4 alkyl), —CONH(C1-C4 alkyl), —CONH2, —NHC(O)(C1-C4 alkyl), —NHC(O)(phenyl), —N(C1-C4 alkyl)C(O)(C1-C4 alkyl), —N(C1-C4 alkyl)C(O)(phenyl), —C(O)C1-C4 alkyl, —C(O)C1-C4 phenyl, —C(O)C1-C4 haloalkyl, —OC(O)C1-C4 alkyl, —SO2(C1-C4 alkyl), —SO2(phenyl), —SO2(C1-C4 haloalkyl), —SO2NH2, —SO2NH(C1-C4 alkyl), —SO2NH(phenyl), —NHSO2(C1-C4 alkyl), —NHSO2(phenyl), and —NHSO2(C1-C4 haloalkyl).
The term “substituted heterocycloalkyloxy” refers to heterocycloalkyloxy wherein the alkyl constituent is substituted (i.e., —O-(substituted heterocycloalkyl)) wherein “substituted heterocycloalkyl” refers to a heterocycloalkyloxy wherein one or more hydrogen atoms are replaced by a substituent independently chosen from:
    • Ra, —ORb, —O(C1-C2 alkyl)O— (e.g., methylenedioxy-), —SRb, guanidine, guanidine wherein one or more of the guanidine hydrogens are replaced with a lower-alkyl group, —NRbRc, halo, cyano, nitro, —CORb, —CO2Rb, —CONRbRc, —OCORb, —OCO2Ra, —OCONRbRc, —NRcCORb, —NRcCO2Ra, —NRcCONRbRc, —CO2Rb, —CONRbRc, —NRcCORb, —SORa, —SO2Ra, —SO2NRbRc, and —NRcSO2Ra,
    • where Ra is chosen from optionally substituted C1-C6 alkyl, optionally substituted aryl, and optionally substituted heteroaryl;
    • Rb is chosen from H, optionally substituted C1-C6 alkyl, optionally substituted aryl, and optionally substituted heteroaryl; and
    • Rc is chosen from hydrogen and optionally substituted C1-C4 alkyl; or
    • Rb and Rc, and the nitrogen to which they are attached, form an optionally substituted heterocycloalkyl group; and
      where each optionally substituted group is unsubstituted or independently substituted with one or more, such as one, two, or three, substituents independently selected from C1-C4 alkyl, aryl, heteroaryl, aryl-C1-C4 alkyl-, heteroaryl-C1-C4 alkyl-, C1-C4 haloalkyl-, —OC1-C4 alkyl, —OC1-C4 alkylphenyl, —C1-C4 alkyl-OH, —OC1-C4 haloalkyl, halo, —OH, —NH2, —C1-C4 alkyl-NH2, —N(C1-C4 alkyl)(C1-C4 alkyl), —NH(C1-C4 alkyl), —N(C1-C4 alkyl)(C1-C4 alkylphenyl), —NH(C1-C4 alkylphenyl), cyano, nitro, oxo (as a substitutent for heteroaryl), —CO2H, —C(O)OC1-C4 alkyl, —CON(C1-C4 alkyl)(C1-C4 alkyl), —CONH(C1-C4 alkyl), —CONH2, —NHC(O)(C1-C4 alkyl), —NHC(O)(phenyl), —N(C1-C4 alkyl)C(O)(C1-C4 alkyl), —N(C1-C4 alkyl)C(O)(phenyl), —C(O)C1-C4 alkyl, —C(O)C1-C4 phenyl, —C(O)C1-C4 haloalkyl, —OC(O)C1-C4 alkyl, —SO2(C1-C4 alkyl), —SO2(phenyl), —SO2(C1-C4 haloalkyl), —SO2NH2, —SO2NH(C1-C4 alkyl), —SO2NH(phenyl), —NHSO2(C1-C4 alkyl), —NHSO2(phenyl), and —NHSO2(C1-C4 haloalkyl).
The term “substituted amino” refers to the group —NHRd or —NRdRd where each Rd is independently chosen from: hydroxy, optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted acyl, aminocarbonyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocycloalkyl, alkoxycarbonyl, sulfinyl and sulfonyl, provided that only one Rd may be hydroxyl, and wherein substituted alkyl, cycloalkyl, aryl, heterocycloalkyl, and heteroaryl refer respectively to alkyl, cycloalkyl, aryl, heterocycloalkyl, and heteroaryl wherein one or more (such as up to 5, for example, up to 3) hydrogen atoms are replaced by a substituent independently chosen from:
    • —Ra, —ORb, —O(C1-C2 alkyl)O— (e.g., methylenedioxy-), —SRb, guanidine, guanidine wherein one or more of the guanidine hydrogens are replaced with a lower-alkyl group, —NRbRc, halo, cyano, nitro, —CORb, —CO2Rb, —CONRbRc, —OCORb, —OCO2Ra, —OCONRbRc, —NRcCORb, —NRcCO2Ra, —NRcCONRbRc, —CO2Rb, —CONRbRc, —NRcCORb, —SORa, —SO2Ra, —SO2NRbRc, and —NRcSO2Ra,
    • where Ra is chosen from optionally substituted C1-C6 alkyl, optionally substituted aryl, and optionally substituted heteroaryl;
    • Rb is chosen from H, optionally substituted C1-C6 alkyl, optionally substituted aryl, and optionally substituted heteroaryl; and
    • Rc is chosen from hydrogen and optionally substituted C1-C4 alkyl; or
    • Rb and Rc, and the nitrogen to which they are attached, form an optionally substituted heterocycloalkyl group; and
    • where each optionally substituted group is unsubstituted or independently substituted with one or more, such as one, two, or three, substituents independently selected from C1-C4 alkyl, aryl, heteroaryl, aryl-C1-C4 alkyl-, heteroaryl-C1-C4 alkyl-, C1-C4 haloalkyl-, —OC1-C4 alkyl, —OC1-C4 alkylphenyl, —C1-C4 alkyl-OH, —OC1-C4 haloalkyl, halo, —OH, —NH2, —C1-C4 alkyl-NH2, —N(C1-C4 alkyl)(C1-C4 alkyl), —NH(C1-C4 alkyl), —N(C1-C4 alkyl)(C1-C4 alkylphenyl), —NH(C1-C4 alkylphenyl), cyano, nitro, oxo (as a substitutent for heteroaryl), —CO2H, —C(O)OC1-C4 alkyl, —CON(C1-C4 alkyl)(C1-C4 alkyl), —CONH(C1-C4 alkyl), —CONH2, —NHC(O)(C1-C4 alkyl), —NHC(O)(phenyl), —N(C1-C4 alkyl)C(O)(C1-C4 alkyl), —N(C1-C4 alkyl)C(O)(phenyl), —C(O)C1-C4 alkyl, —C(O)C1-C4 phenyl, —C(O)C1-C4 haloalkyl, —OC(O)C1-C4 alkyl, —SO2(C1-C4 alkyl), —SO2(phenyl), —SO2(C1-C4 haloalkyl), —SO2NH2, —SO2NH(C1-C4 alkyl), —SO2NH(phenyl), —NHSO2(C1-C4 alkyl), —NHSO2(phenyl), and —NHSO2(C1-C4haloalkyl); and
    • wherein optionally substituted acyl, aminocarbonyl, alkoxycarbonyl, sulfinyl and sulfonyl are as defined herein.
The term “substituted amino” also refers to N-oxides of the groups —NHRd, and NRdRd each as described above. N-oxides can be prepared by treatment of the corresponding amino group with, for example, hydrogen peroxide or m-chloroperoxybenzoic acid. The person skilled in the art is familiar with reaction conditions for carrying out the N-oxidation.
Compounds described herein include, but are not limited to, their optical isomers, racemates, and other mixtures thereof. In those situations, the single enantiomers or diastereomers, i.e., optically active forms, can be obtained by asymmetric synthesis or by resolution of the racemates. Resolution of the racemates can be accomplished, for example, by conventional methods such as crystallization in the presence of a resolving agent, or chromatography, using, for example a chiral high-pressure liquid chromatography (HPLC) column. In addition, such compounds include Z- and E-forms (or cis- and trans-forms) of compounds with carbon-carbon double bonds. Where compounds described herein exist in various tautomeric forms, chemical entities include all tautomeric forms of the compound. Such compounds also include crystal forms including polymorphs and clathrates.
Chemical entities include, but are not limited to compounds described herein and all pharmaceutically acceptable forms thereof. Pharmaceutically acceptable forms of the compounds recited herein include pharmaceutically acceptable salts, solvates, chelates, non-covalent complexes, prodrugs, and mixtures thereof. In some embodiments, the compounds described herein are in the form of pharmaceutically acceptable salts. Hence, the terms “chemical entity” and “chemical entities” also encompass pharmaceutically acceptable salts, solvates, chelates, non-covalent complexes, prodrugs, and mixtures.
“Pharmaceutically acceptable salts” include, but are not limited to salts with inorganic acids, such as hydrochlorate, phosphate, diphosphate, hydrobromate, sulfate, sulfinate, nitrate, and like salts; as well as salts with an organic acid, such as malate, maleate, fumarate, tartrate, succinate, citrate, acetate, lactate, methanesulfonate, p-toluenesulfonate, 2-hydroxyethylsulfonate, benzoate, salicylate, stearate, and alkanoate such as acetate, HOOC—(CH2)n—COOH where n is 0-4, and like salts. Similarly, pharmaceutically acceptable cations include, but are not limited to sodium, potassium, calcium, aluminum, lithium, and ammonium.
In addition, if the compounds described herein are obtained as an acid addition salt, the free base can be obtained by basifying a solution of the acid salt. Conversely, if the product is a free base, an addition salt, particularly a pharmaceutically acceptable addition salt, may be produced by dissolving the free base in a suitable organic solvent and treating the solution with an acid, in accordance with conventional procedures for preparing acid addition salts from base compounds. Those skilled in the art will recognize various synthetic methodologies that may be used to prepare non-toxic pharmaceutically acceptable addition salts.
As noted above, prodrugs also fall within the scope of chemical entities, for example ester or amide derivatives of the compounds described herein. The term “prodrugs” includes any compounds that become compounds described herein when administered to a patient, e.g., upon metabolic processing of the prodrug. Examples of prodrugs include, but are not limited to, acetate, formate, and benzoate and like derivatives of functional groups (such as alcohol or amine groups) in the compounds of described herein.
The term “solvate” refers to the chemical entity formed by the interaction of a solvent and a compound. Suitable solvates are pharmaceutically acceptable solvates, such as hydrates, including monohydrates and hemi-hydrates.
The term “chelate” refers to the chemical entity formed by the coordination of a compound to a metal ion at two (or more) points.
The term “non-covalent complex” refers to the chemical entity formed by the interaction of a compound and another molecule wherein a covalent bond is not formed between the compound and the molecule. For example, complexation can occur through van der Waals interactions, hydrogen bonding, and electrostatic interactions (also called ionic bonding).
The term “hydrogen bond” refers to a form of association between an electronegative atom (also known as a hydrogen bond acceptor) and a hydrogen atom attached to a second, relatively electronegative atom (also known as a hydrogen bond donor). Suitable hydrogen bond donor and acceptors are well understood in medicinal chemistry (G. C. Pimentel and A. L. McClellan, The Hydrogen Bond, Freeman, San Francisco, 1960; R. Taylor and O. Kennard, “Hydrogen Bond Geometry in Organic Crystals”, Accounts of Chemical Research, 17, pp. 320-326 (1984)).
“Hydrogen bond acceptor” refers to a group comprising an oxygen or nitrogen, especially an oxygen or nitrogen that is sp2-hybridized, an ether oxygen, or the oxygen of a sulfoxide or N-oxide.
The term “hydrogen bond donor” refers to an oxygen, nitrogen, or heteroaromatic carbon that bears a hydrogen group containing a ring nitrogen or a heteroaryl group containing a ring nitrogen.
As used herein the terms “group”, “radical” or “fragment” are synonymous and are intended to indicate functional groups or fragments of molecules attachable to a bond or other fragments of molecules.
The term “active agent” is used to indicate a chemical entity which has biological activity. In some embodiments, an “active agent” is a compound having pharmaceutical utility. For example an active agent may be an anti-cancer therapeutic.
The term “therapeutically effective amount” of a chemical entity described herein means an amount effective, when administered to a human or non-human patient, to provide a therapeutic benefit such as amelioration of symptoms, slowing of disease progression, or prevention of disease e.g., a therapeutically effective amount may be an amount sufficient to decrease the symptoms of a disease responsive to inhibition of Syk activity. In some embodiments, a therapeutically effective amount is an amount sufficient to reduce cancer symptoms, the symptoms of an allergic disorder, the symptoms of an autoimmune and/or inflammatory disease, or the symptoms of an acute inflammatory reaction. In some embodiments a therapeutically effective amount is an amount sufficient to decrease the number of detectable cancerous cells in an organism, detectably slow, or stop the growth of a cancerous tumor. In some embodiments, a therapeutically effective amount is an amount sufficient to shrink a cancerous tumor. In some circumstances a patient suffering from cancer may not present symptoms of being affected. In some embodiments, a therapeutically effective amount of a chemical entity is an amount sufficient to prevent a significant increase or significantly reduce the detectable level of cancerous cells or cancer markers in the patient's blood, serum, or tissues. In methods described herein for treating allergic disorders and/or autoimmune and/or inflammatory diseases and/or acute inflammatory reactions, a therapeutically effective amount may also be an amount sufficient, when administered to a patient, to detectably slow progression of the disease, or prevent the patient to whom the chemical entity is given from presenting symptoms of the allergic disorders and/or autoimmune and/or inflammatory disease, and/or acute inflammatory response. In some methods described herein for treating allergic disorders and/or autoimmune and/or inflammatory diseases and/or acute inflammatory reactions, a therapeutically effective amount may also be an amount sufficient to produce a detectable decrease in the amount of a marker protein or cell type in the patient's blood or serum. For example, in some embodiments a therapeutically effective amount is an amount of a chemical entity described herein sufficient to significantly decrease the activity of B-cells. In another example, in some embodiments a therapeutically effective amount is an amount of a chemical entity described herein sufficient to significantly decrease the number of B-cells. In another example, in some embodiments a therapeutically effective amount is an amount of a chemical entity described herein sufficient to decrease the level of anti-acetylcholine receptor antibody in a patient's blood with the disease myasthenia gravis.
The term “inhibition” indicates a significant decrease in the baseline activity of a biological activity or process. “Inhibition of Syk activity” refers to a decrease in Syk activity as a direct or indirect response to the presence of at least one chemical entity described herein, relative to the activity of Syk in the absence of the at least one chemical entity. The decrease in activity may be due to the direct interaction of the compound with Syk, or due to the interaction of the chemical entity(ies) described herein with one or more other factors that in turn affect Syk activity. For example, the presence of the chemical entity(ies) may decrease Syk activity by directly binding to the Syk, by causing (directly or indirectly) another factor to decrease Syk activity, or by (directly or indirectly) decreasing the amount of Syk present in the cell or organism.
Inhibition of Syk activity also refers to observable inhibition of Syk activity in a standard biochemical assay for Syk activity, such as the ATP hydrolysis assay described below. In some embodiments, the chemical entity described herein has an IC50 value less than or equal to 1 micromolar. In some embodiments, the chemical entity has an IC50 value less than or equal to less than 100 nanomolar. In some embodiments, the chemical entity has an IC50 value less than or equal to 10 nanomolar.
“Inhibition of B-cell activity” refers to a decrease in B-cell activity as a direct or indirect response to the presence of at least one chemical entity described herein, relative to the activity of B-cells in the absence of the at least one chemical entity. The decrease in activity may be due to the direct interaction of the compound with Syk or with one or more other factors that in turn affect B-cell activity.
Inhibition of B-cell activity also refers to observable inhibition of CD86 expression in a standard assay such as the assay described below. In some embodiments, the chemical entity described herein has an IC50 value less than or equal to 10 micromolar. In some embodiments, the chemical entity has an IC50 value less than or equal to less than 1 micromolar. In some embodiments, the chemical entity has an IC50 value less than or equal to 500 nanomolar.
“B cell activity” also includes activation, redistribution, reorganization, or capping of one or more various B cell membrane receptors, or membrane-bound immunoglobulins, e.g, IgM, IgG, and IgD. Most B cells also have membrane receptors for Fc portion of IgG in the form of either antigen-antibody complexes or aggregated IgG. B cells also carry membrane receptors for the activated components of complement, e.g., C3b, C3d, C4, and C1q. These various membrane receptors and membrane-bound immunoglobulins have membrane mobility and can undergo redistribution and capping that can initiate signal transduction.
B cell activity also includes the synthesis or production of antibodies or immunoglobulins. Immunoglobulins are synthesized by the B cell series and have common structural features and structural units. Five immunoglobulin classes, i.e., IgG, IgA, IgM, IgD, and IgE, are recognized on the basis of structural differences of their heavy chains including the amino acid sequence and length of the polypeptide chain. Antibodies to a given antigen may be detected in all or several classes of immunoglobulins or may be restricted to a single class or subclass of immunoglobulin. Autoantibodies or autoimmune antibodies may likewise belong to one or several classes of immunoglobulins. For example, rheumatoid factors (antibodies to IgG) are most often recognized as an IgM immunoglobulin, but can also consist of IgG or IgA.
In addition, B cell activity also is intended to include a series of events leading to B cell clonal expansion (proliferation) from precursor B lymphocytes and differentiation into antibody-synthesizing plasma cells which takes place in conjunction with antigen-binding and with cytokine signals from other cells.
“Inhibition of B-cell proliferation” refers to inhibition of proliferation of abnormal B-cells, such as cancerous B-cells, e.g. lymphoma B-cells and/or inhibition of normal, non-diseased B-cells. The term “inhibition of B-cell proliferation” indicates any significant decrease in the number of B-cells, either in vitro or in vivo. Thus an inhibition of B-cell proliferation in vitro would be any significant decrease in the number of B-cells in an in vitro sample contacted with at least one chemical entity described herein as compared to a matched sample not contacted with the chemical entity(ies).
Inhibition of B-cell proliferation also refers to observable inhibition of B-cell proliferation in a standard thymidine incorporation assay for B-cell proliferation, such as the assay described herein. In some embodiments, the chemical entity has an IC50 value less than or equal to 10 micromolar. In some embodiments, the chemical entity has an IC50 value less than or equal to less than 1 micromolar. In some embodiments, the chemical entity has an IC50 value less than or equal to 500 nanomolar.
An “allergy” or “allergic disorder” refers to acquired hypersensitivity to a substance (allergen). Allergic conditions include eczema, allergic rhinitis or coryza, hay fever, bronchial asthma, urticaria (hives) and food allergies, and other atopic conditions.
“Asthma” refers to a disorder of the respiratory system characterized by inflammation, narrowing of the airways and increased reactivity of the airways to inhaled agents. Asthma is frequently, although not exclusively associated with atopic or allergic symptoms.
By “significant” is meant any detectable change that is statistically significant in a standard parametric test of statistical significance such as Student's T-test, where p<0.05.
A “disease responsive to inhibition of Syk activity” is a disease in which inhibiting Syk kinase provides a therapeutic benefit such as an amelioration of symptoms, decrease in disease progression, prevention or delay of disease onset, or inhibition of aberrant activity of certain cell-types (monocytes, B-cells, and mast cells).
“Treatment or treating means any treatment of a disease in a patient, including:
    • a) preventing the disease, that is, causing the clinical symptoms of the disease not to develop;
    • b) inhibiting the disease;
    • c) slowing or arresting the development of clinical symptoms; and/or
    • d) relieving the disease, that is, causing the regression of clinical symptoms.
“Patient” refers to an animal, such as a mammal, that has been or will be the object of treatment, observation or experiment. The methods described herein may be useful in both human therapy and veterinary applications. In some embodiments, the patient is a mammal; in some embodiments the patient is human; and in some embodiments the patient is chosen from cats and dogs.
Provided is at least one chemical entity chosen from compounds of Formula I
Figure US08697699-20140415-C00004
and pharmaceutically acceptable salts thereof, wherein:
    • W is selected from optionally substituted aryl and optionally substituted heteroaryl;
    • A is selected from optionally substituted alkyl, optionally substituted alkoxy, optionally substituted amino, carboxy, cyano, halo, hydrogen, hydroxy and nitro;
    • X is selected from CH and N;
    • Q is selected from CO and SO2;
    • Y is selected from hydrogen, optionally substituted alkyl; optionally substituted cycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, and optionally substituted heterocycloalkyl;
    • Z is selected from hydrogen, optionally substituted alkyl; optionally substituted cycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, and optionally substituted heterocycloalkyl; or
    • Z taken together with Y, and the nitrogen to which they are bound, form an optionally substituted 5- to 13-membered nitrogen-containing ring which optionally includes one or two additional heteroatoms chosen from O and S;
    • provided that if W is 4-(morpholino)phenyl, A is hydrogen, X is CH, Q is CO and Y is hydrogen, then Z is not hydroxyethyl;
    • provided that if W is p-chlorophenyl or 4-(p-tolylsulfonamido)phenyl, A is hydrogen, X is CH, Q is CO and Y is hydrogen, then Z is not N,N-dimethylaminoethyl; and
    • provided that if W is 4-(morpholinecarbonyl)phenyl, A is hydrogen, X is CH, Q is CO and Y is hydrogen, then Z is not 3-chlorobenzyl or 4-t-butylphenyl.
Provided is at least one chemical entity selected from compounds of Formula I
Figure US08697699-20140415-C00005
and pharmaceutically acceptable salts thereof, wherein:
    • W is selected from optionally substituted aryl and optionally substituted heteroaryl;
    • A is selected from optionally substituted alkyl, optionally substituted alkoxy, optionally substituted amino, carboxy, cyano, halo, hydrogen, hydroxy and nitro;
    • X is selected from CH and N;
    • Q is selected from CO and SO2;
    • Y is selected from hydrogen, optionally substituted alkyl; optionally substituted cycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, and optionally substituted heterocycloalkyl;
    • Z is selected from hydrogen, optionally substituted alkyl; optionally substituted cycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, and optionally substituted heterocycloalkyl; or
    • Z taken together with Y, and the nitrogen to which they are bound, form a 5- to 13-membered nitrogen-containing ring;
    • provided that if W is 4-(morpholino)phenyl, A is hydrogen, X is CH, Q is CO and Y is hydrogen, then Z is not hydroxyethyl;
    • provided that if W is p-chlorophenyl or 4-(p-tolylsulfonamido)phenyl, A is hydrogen, X is CH, Q is CO and Y is hydrogen, then Z is not N,N-dimethylaminoethyl; and
    • provided that if W is 4-(morpholinecarbonyl)phenyl, A is hydrogen, X is CH, Q is CO and Y is hydrogen, then Z is not 3-chlorobenzyl or 4-t-butylphenyl.
In some embodiments, Q is SO2. In some embodiments, Q is C(O).
In some embodiments, W is optionally substituted aryl. In some embodiments, W is optionally substituted heteroaryl.
In some embodiments, A is selected from halo, optionally substituted lower alkyl, and hydrogen. In some embodiments, A is selected from halo, lower alkyl, and hydrogen. In some embodiments, A is hydrogen.
In some embodiments, X is CH. In some embodiments, X is N.
In some embodiments, Y is selected from optionally substituted lower alkyl and hydrogen. In some embodiments, Y is selected from lower alkyl and hydrogen. In some embodiments, Y is hydrogen.
In some embodiments, Y and Z are taken together with the nitrogen to which they are bound to form a 5- to 13-membered nitrogen-containing ring. In some embodiments, Y and Z are taken together with the nitrogen to which they are bound to form a 5- to 13-membered nitrogen-containing ring selected from optionally substituted piperazinyl, optionally substituted piperidinyl and optionally substituted diazepanyl. In some embodiments, Z taken together with Y, and the nitrogen to which they are bound, form an optionally substituted 5- to 13-membered nitrogen-containing ring which also contains an additional oxygen heteroatom. In some embodiments, Z taken together with Y, and the nitrogen to which they are bound, form an optionally substituted morpholinyl ring.
In some embodiments, Z is selected from hydrogen, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted cycloalkyl and optionally substituted heterocycloakyl. In some embodiments, Z is selected from optionally substituted phenyl, optionally substituted piperidinyl, optionally substituted benzodiazolyl, optionally substituted pyridinyl and optionally substituted cyclohexyl.
In some embodiments, Z is selected from hydrogen and lower alkyl that is optionally substituted with a group chosen from amino, (lower alkyl)amino, di-(lower alkyl)amino, hydroxy, and lower alkoxy. In some embodiments, Z is selected from hydrogen.
In some embodiments, Z is represented by the following structure:
Figure US08697699-20140415-C00006
    • wherein
    • R6, R7, R8, R9 and R10 are each independently selected from optionally substituted acyl, optionally substituted alkoxy, optionally substituted alkoxycarbonyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted amino, optionally substituted aminocarbonyl, optionally substituted aryl, optionally substituted aryloxy, carboxy, cyano, optionally substituted cycloalkyl, optionally substituted cycloalkyloxy, halo, optionally substituted heteroaryl, optionally substituted heteroaryloxy, optionally substituted heterocycloalkyl, optionally substituted heterocycloalkyloxy, hydrogen, hydroxy, nitro, phosphono, sulfanyl, sulfinyl, sulfonyl, and optionally substituted thiocarbonyl; or
    • two adjacent substituents selected from R6, R7, R8, R9 and R10 are taken together with the carbons to which they are bound to form a fused 5 to 7-membered ring chosen from optionally substituted cycloalkyl rings, optionally substituted aryl rings, optionally substituted heteroaryl rings and optionally substituted heterocyclic rings; and
    • V is selected from C and N, wherein if V is N, then R9 is absent.
In some embodiments, Y and Z are both hydrogen.
In some embodiments, V is C. In some embodiments, V is N.
In some embodiments, R6, R7, R8, R9 and R10 are each independently selected from optionally substituted C1-C6alkoxy, optionally substituted C1-C6 alkyl, optionally substituted C1-C6 alkylcarbonyl, optionally substituted aminocarbonyl, optionally substituted aryl, carboxy, cyano, optionally substituted C1-C6alkoxycarbonyl, halo, optionally substituted heteroaryl, hydrogen, hydroxy, and sulfonyl. In some embodiments, R6, R7, R9 and R10 are each hydrogen.
In some embodiments, R8 is selected from optionally substituted C1-C6 alkyl, optionally substituted C1-C6alkoxy, optionally substituted C1-C6alkoxycarbonyl, optionally substituted aminocarbonyl, carboxy, cyano, halo, optionally substituted heteroaryl, optionally substituted heterocycloalkyl, hydroxyl and sulfonyl. In some embodiments, R8 is carboxy. In some embodiments, R8 is —C(O)—NH(lower alkyl).
Also provided is at least one chemical entity chosen from compounds of Formula II:
Figure US08697699-20140415-C00007
and pharmaceutically acceptable salts thereof,
    • wherein A, X, Y, and Z are as described for compounds of Formula I and wherein
    • R1, R2, R3, R4 and R5 are each independently selected from optionally substituted acyl, optionally substituted alkoxy, optionally substituted alkoxycarbonyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted amino, optionally substituted aminocarbonyl, optionally substituted aryl, optionally substituted aryloxy, carboxy, cyano, optionally substituted cycloalkyl, optionally substituted cycloalkyloxy, halo, optionally substituted heteroaryl, optionally substituted heteroaryloxy, optionally substituted heterocycloalkyl, optionally substituted heterocycloalkyloxy, hydrogen, hydroxy, nitro, phosphono, sulfanyl, sulfinyl, sulfonyl, and optionally substituted thiocarbonyl; or
    • two adjacent substituents selected from R1, R2, R3, R4 and R5 are taken together with the carbons to which they are bound to form a fused 5 to 7-membered ring chosen from optionally substituted cycloalkyl rings, optionally substituted aryl rings, optionally substituted heteroaryl rings and optionally substituted heterocyclic rings.
In some embodiments, R1, R2, R3, R4 and R5 are each independently selected from optionally substituted C1-C6 alkoxy, optionally substituted C1-C6 alkyl, optionally substituted aminocarbonyl, optionally substituted heterocycloalkyl and hydrogen. In some embodiments, R1, R2 and R5 are hydrogen. In some embodiments, R3 and R4 are each independently selected from optionally substituted C1-C6 alkoxy, optionally substituted C1-C6 alkyl, and optionally substituted heterocycloalkyl. In some embodiments, R3 and R4 are both independently optionally substituted C1-C6 alkoxy. In some embodiments, R3 and R4 are both methoxy.
Also provided is at least one chemical entity chosen from compounds of Formula III:
Figure US08697699-20140415-C00008
and pharmaceutically acceptable salts thereof,
wherein A, X, Y, and Z are as described for compounds of Formula I and wherein R1, R2, R3, R4 and R5 are as described for compounds of Formula II.
Also provided is at least one chemical entity chosen from
  • 3-{8-[(3,4-Dimethoxyphenyl)-amino]imidazo[1,2-a]pyrazin-6-yl}-N-(piperidin-4-yl)benzamide;
  • 6-[3-(2,3-Dihydro-1H-indol-1-ylcarbonyl)phenyl]-N-(3,4-Dimethoxyphenyl)-imidazo[1,2-a]pyrazin-8-amine;
  • 4-({3-[8-(1H-Indazol-6-ylamino)-imidazo[1,2-a]pyrazin-6-yl]benzene}amido)benzoic acid;
  • 4-[(3-{8-[(3,4-Dimethoxyphenyl)amino]-imidazo[1,2-a]pyrazin-6-yl}benzene)-amido]cyclohexane-1-carboxylic acid;
  • 4-{1-[(3-{8-[(3,4-Dimethoxyphenyl)-amino]imidazo[1,2-a]pyrazin-6-yl}-phenyl)formamido]-2-methylpropan-2-yl}benzoic acid;
  • 2-(Morpholin-4-yl)ethyl 4-[(3-{8-[(3,4-Dimethoxyphenyl)amino]imidazo[1,2-a]pyrazin-6-yl}benzene)amido]benzoate;
  • 2-{4-[(3-{8-[(3,4-Dimethoxyphenyl)-amino]imidazo[1,2-a]pyrazin-6-yl}-phenyl)carbonyl]piperazin-1-yl}ethan-1-ol;
  • N-(3,4-Dimethoxyphenyl)-6-{3-[(4-methylpiperazin-1-yl)carbonyl]phenyl}-imidazo[1,2-a]pyrazin-8-amine;
  • N-(3,4-Dimethoxyphenyl)-6-[3-(piperazin-1-ylcarbonyl)phenyl]-imidazo[1,2-a]pyrazin-8-amine;
  • 1-[(3-{8-[(3,4-Dimethoxyphenyl)amino]-imidazo[1,2-a]pyrazin-6-yl}phenyl)-carbonyl]piperidine-4-carboxylic acid;
  • 3-{8-[(3,4-Dimethoxyphenyl)amino]-imidazo[1,2-a]pyrazin-6-yl}-N,N-dimethylbenzamide;
  • N-(3,4-Dimethoxyphenyl)-6-[3-(1,2,3,4-tetrahydroisoquinolin-2-ylcarbonyl)-phenyl]imidazo[1,2-a]pyrazin-8-amine;
  • 3-{8-[(3,4-Dimethoxyphenyl)amino]-imidazo[1,2-a]pyrazin-6-yl}-N-(1-methylpiperidin-4-yl)benzamide;
  • 4-[(3-{8-[(3,4-Dimethoxyphenyl)amino]-imidazo[1,2-a]pyrazin-6-yl}phenyl)-carbonyl]piperazin-2-one;
  • 4-{8-[(3,4-Dimethoxyphenyl)amino]-imidazo[1,2-a]pyrazin-6-yl}-N-[2-(pyridin-4-yl)ethyl]benzamide;
  • 3-{8-[(3,4-Dimethoxyphenyl)amino]-imidazo[1,2-a]pyrazin-6-yl}-N-methylbenzamide;
  • 3-{8-[(3,4-Dimethoxyphenyl)amino]imidazo[1,2-a]pyrazin-6-yl}-N-(pyridin-4-ylmethyl)-benzamide;
  • 4-{[(3-{8-[(3,4-Dimethoxyphenyl)amino]-imidazo[1,2-a]pyrazin-6-yl}phenyl)-formamido]methyl}benzoic acid;
  • 6-[3-(1,4-Diazepan-1-ylcarbonyl)-phenyl]-N-(3,4-dimethoxyphenyl)-imidazo[1,2-a]pyrazin-8-amine;
  • 3-{8-[(3,4-Dimethoxyphenyl)amino]imidazo[1,2-a]pyrazin-6-yl}-N-[(6-hydroxypyridin-3-yl)methyl]benzamide;
  • 2-{4-[(3-{8-[(3,4-Dimethoxyphenyl)-amino]imidazo[1,2-a]pyrazin-6-yl}-phenyl)carbonyl]piperazin-1-yl}acetic acid;
  • 1-[(3-{8-[(3,4-Dimethoxyphenyl)amino]-imidazo[1,2-a]pyrazin-6-yl}phenyl)-carbonyl]piperidin-4-ol;
  • 5-{8-[(3,4-dimethoxyphenyl)amino]-imidazo[1,2-a]pyrazin-6-yl}-N-[4-(1H-pyrazol-4-yl)phenyl]pyridine-3-carboxamide;
  • N-(1H-1,3-Benzodiazol-6-yl)-3-{8-[(3,4-Dimethoxyphenyl)amino]imidazo[1,2-a]pyrazin-6-yl}benzamide;
  • 4-[(3-{8-[(3,4-Dimethoxyphenyl)-amino]imidazo[1,2-a]pyrazin-6-yl}benzene)amido]-2-methoxybenzoic acid;
  • 4-[(3-{8-[(3,4-Dimethoxyphenyl)amino]imidazo[1,2-a]pyrazin-6-yl}benzene)amido]-2-fluorobenzoic acid;
  • Benzyl 4-[(3-{8-[(3,4-Dimethoxyphenyl)-amino]imidazo[1,2-a]pyrazin-6-yl}-benzene)amido]benzoate;
  • N-(1H-1,2,3-Benzotriazol-5-yl)-3-{8-[(3,4-Dimethoxyphenyl)amino]-imidazo[1,2-a]pyrazin-6-yl}benzamide;
  • 4-[(3-{8-[(3,4-Dimethoxyphenyl)amino]-imidazo[1,2-a]pyrazin-6-yl}-2-methyl-benzene)amido]benzoic acid;
  • 3-[(4-{8-[(3,4-Dimethoxyphenyl)amino]-imidazo[1,2-a]pyrazin-6-yl}benzene)-amido]benzoic acid;
  • 4-[3-{8-[(3,4-Dimethoxyphenyl)amino]-imidazo[1,2-a]pyrazin-6-yl}benzene)-amido]-3-methylbenzoic acid;
  • 3-{8-[(3,4-Dimethoxyphenyl)amino]-imidazo[1,2-a]pyrazin-6-yl}-N-[3-(methylsulfamoyl)phenyl]benzamide;
  • 3-{8-[(3,4-Dimethoxyphenyl)amino]-imidazo[1,2-a]pyrazin-6-yl}-N-phenylbenzamide;
  • 3-{8-[(3,4-Dimethoxyphenyl)amino]-imidazo[1,2-a]pyrazin-6-yl}-N-(1H-indazol-5-yl)benzamide;
  • 3-{8-[(3,4-Dimethoxyphenyl)amino]-imidazo[1,2-a]pyrazin-6-yl}-N-(1H-indazol-6-yl)-benzamide;
  • 3-[(3-{8-[(3,4-Dimethoxyphenyl)amino]-imidazo[1,2-a]pyrazin-6-yl}benzene)-amido]benzoic acid;
  • 4-[(3-{8-[(3,4-Dimethoxyphenyl)amino]-imidazo[1,2-a]pyrazin-6-yl}benzene)amido]-2-methylbenzoic acid;
  • N-(1H-1,3-benzodiazol-5-yl)-3-(8-{[4-(morpholin-4-yl)phenyl]amino}-imidazo[1,2-a]pyrazin-6-yl)benzamide;
  • 3-{8-[(3,4-dimethoxyphenyl)amino]-imidazo[1,2-a]pyrazin-6-yl}-N-[3-methyl-4-(1H-pyrazol-4-yl)phenyl]benzamide;
  • 3-{8-[(3,4-dimethoxyphenyl)amino]-imidazo[1,2-a]pyrazin-6-yl}-N-[2-methyl-4-(1H-pyrazol-4-yl)phenyl]benzamide;
  • 4-{3-[8-(3,4-Dimethoxy-phenylamino)-imidazo[1,2-a]pyrazin-6-yl]-benzoylamino}-benzoic acid;
  • 3-{8-[(3-Methoxy-4-methylphenyl)amino]imidazo[1,2-a]pyrazin-6-yl}-N-[4-(1H-pyrazol-4-yl)phenyl]benzamide;
  • 5-(3-(8-(3,4-Dimethoxyphenylamino)-imidazo[1,2-a]pyrazin-6-yl)benzamido)-N-methylpicolinamide;
  • 3-{8-[(3,4-Dimethoxyphenyl)amino]-imidazo[1,2-a]pyrazin-6-yl}-N-(4-acetylphenyl)benzamide;
  • 3-{8-[(3,4-Dimethoxyphenyl)amino]-imidazo[1,2-a]pyrazin-6-yl}-N-[4-(methylcarbamoyl)phenyl]benzamide;
  • N-(4-Cyanophenyl)-3-{8-[(3,4-Dimethoxyphenyl)amino]imidazo[1,2-a]pyrazin-6-yl}benzamide;
  • Ethyl 4-[(3-{8-[(3,4-Dimethoxyphenyl)-amino]imidazo[1,2-a]pyrazin-6-yl}benzene)amido]benzoate;
  • 3-{8-[(3,4-Dimethoxyphenyl)amino]-imidazo[1,2-a]pyrazin-6-yl}-N-[4-(methanesulfonylcarbamoyl)phenyl]-benzamide;
  • 3-{8-[(3,4-Dimethoxyphenyl)amino]-imidazo[1,2-a]pyrazin-6-yl}-N-[4-(1H-pyrazol-4-yl)phenyl]benzamide;
  • 3-{8-[(3,4-Dimethoxyphenyl)amino]-imidazo[1,2-a]pyrazin-6-yl}-N-[4-(1H-1,2,3,4-tetrazol-5-yl)phenyl]benzamide;
  • N-{4-[(Dimethylamino)methyl]phenyl}-3-(8-{[4-(morpholin-4-yl)phenyl]amino}-imidazo[1,2-a]pyrazin-6-yl)benzamide;
  • 3-{8-[(3,4-Dimethoxyphenyl)amino]-imidazo[1,2-a]pyrazin-6-yl}-N-[4-(piperazin-1-yl)phenyl]benzamide;
  • 3-(8-{[4-(Morpholin-4-yl)phenyl]-amino}imidazo[1,2-a]pyrazin-6-yl)-N-[4-(1H-pyrazol-4-yl)phenyl]benzamide;
  • 3-{8-[(3,4-Dimethoxyphenyl)amino]-imidazo[1,2-a]pyrazin-6-yl}-N-[4-(1-hydroxyethyl)phenyl]benzamide;
  • 3-{8-[(3,4-Dimethoxyphenyl)amino]-imidazo[1,2-a]pyrazin-6-yl}-N-[4-(1H-imidazol-2-yl)phenyl]benzamide;
  • 2-{4-[(3-{8-[(3,4-Dimethoxyphenyl)-amino]imidazo[1,2-a]pyrazin-6-yl}benzene)amido]phenyl}acetic acid;
  • 3-{8-[(3,4-Dimethoxyphenyl)amino]-imidazo[1,2-a]pyrazin-6-yl}-N-[4-(methylsulfamoyl)phenyl]benzamide;
  • N-{4-[(1R)-1-Aminoethyl]phenyl}-3-{8-[(3,4-dimethoxyphenyl)amino]-imidazo[1,2-a]pyrazin-6-yl}benzamide;
  • 3-[8-({4-[(4-ethylpiperazin-1-yl)methyl]-phenyl}amino)imidazo[1,2-a]pyrazin-6-yl]-N-[4-(1H-pyrazol-4-yl)phenyl]-benzamide;
  • 3-{8-[(3,4-Dimethoxyphenyl)amino]-imidazo[1,2-a]pyrazin-6-yl}-N-[4-(1H-pyrazol-5-yl)phenyl]benzamide;
  • 5-[(3-{8-[(3,4-Dimethoxyphenyl)amino]-imidazo[1,2-a]pyrazin-6-yl}benzene)-amido]pyridine-2-carboxylic acid;
  • 3-{8-[(3,4-Dimethoxyphenyl)amino]-imidazo[1,2-a]pyrazin-6-yl}-N-{4-[(methylcarbamoyl)methyl]phenyl}benzamide;
  • N-(4-Carbamoylphenyl)-3-{8-[(3,4-dimethoxyphenyl)amino]imidazo[1,2-a]pyrazin-6-yl}benzamide;
  • 3-{8-[(3,4-Dimethoxyphenyl)amino]imidazo[1,2-a]pyrazin-6-yl}-N-(4-fluorophenyl)benzamide;
  • Methyl 4-[(3-{8-[(3,4-Dimethoxyphenyl)-amino]imidazo[1,2-a]pyrazin-6-yl}benzene)amido]benzoate;
  • N-{4-[(1S)-1-Aminoethyl]phenyl}-3-{8-[(3,4-dimethoxyphenyl)amino]-imidazo[1,2-a]pyrazin-6-yl}benzamide;
  • 3-{8-[(3,4-Dimethoxyphenyl)amino]-imidazo[1,2-a]pyrazin-6-yl}-N-(4-methylphenyl)benzamide;
  • 3-{8-[(3,4-Dimethoxyphenyl)amino]-imidazo[1,2-a]pyrazin-6-yl}-N-(6-hydroxypyridin-3-yl)benzamide;
  • N-{4-[(methylamino)methyl]phenyl}-3-(8-{[4-(morpholin-4-yl)phenyl]amino}imidazo[1,2-a]pyrazin-6-yl)benzamide;
  • 4-{[3-(8-{[4-(morpholin-4-yl)phenyl]-amino}imidazo[1,2-a]pyrazin-6-yl)benzene]amido}benzoic acid;
  • 3-[8-({4-[2-(dimethylamino)ethoxy]-3-methoxyphenyl}amino)imidazo[1,2-a]pyrazin-6-yl]-N-[4-(1H-pyrazol-4-yl)phenyl]benzamide;
  • N-[4-(hydroxycarbamoyl)phenyl]-3-(8-{[4-(morpholin-4-yl)phenyl]amino}-imidazo[1,2-a]pyrazin-6-yl)benzamide;
  • 3-(8-{[4-(morpholin-4-yl)phenyl]amino}-imidazo[1,2-a]pyrazin-6-yl)-N-[4-(1H-1,2,4-triazol-3-yl)phenyl]benzamide;
  • 3-(8-{[3-methoxy-4-(morpholin-4-yl)-phenyl]amino}imidazo[1,2-a]pyrazin-6-yl)-N-[4-(1H-pyrazol-4-yl)phenyl]-benzamide;
  • 3-(8-{[3-methoxy-4-(morpholin-4-ylcarbonyl)phenyl]amino}imidazo[1,2-a]pyrazin-6-yl)-N-[4-(1H-pyrazol-4-yl)phenyl]benzamide;
  • N-[4-(1H-imidazol-4-yl)phenyl]-3-(8-{([4-(morpholin-4-yl)phenyl]amino}-imidazo[1,2-a]pyrazin-6-yl)benzamide;
  • 3-{8-[(3,4-diethoxyphenyl)amino]-imidazo[1,2-a]pyrazin-6-yl}-N-[4-(1H-pyrazol-4-yl)phenyl]benzamide;
  • 3-{8-[(3-ethoxy-4-methoxyphenyl)-amino]imidazo[1,2-a]pyrazin-6-yl}-N-[4-(methylcarbamoyl)phenyl]benzamide;
  • 3-{8-[(4-ethoxy-3-methoxyphenyl)-amino]imidazo[1,2-a]pyrazin-6-yl}-N-[4-(methylcarbamoyl)phenyl]benzamide;
  • 3-{8-[(4-methoxy-3-methylphenyl)-amino]imidazo[1,2-a]pyrazin-6-yl}-N-[4-(methylcarbamoyl)phenyl]benzamide;
  • 4-{8-[(3,4-Dimethoxyphenyl)amino]-imidazo[1,2-a]pyrazin-6-yl}-N-[2-(pyridin-4-yl)ethyl]benzamide;
  • 4-[(3-{8-[(3,4-Dimethoxyphenyl)amino]-imidazo[1,2-a]pyrazin-6-yl}benzene)-sulfonamido]benzoic acid;
  • 4-{8-[(3,4-Dimethoxyphenyl)amino]-imidazo[1,2-a]pyrazin-6-yl}-N-(pyridin-3-ylmethyl)benzamide;
  • 3-{8-[(6-methoxypyridin-3-yl)amino]-imidazo[1,2-a]pyrazin-6-yl}-N-[4-(methylcarbamoyl)phenyl]benzamide; and
  • 3-{(8-[(1-ethyl-1H-pyrazol-4-yl)amino]-imidazo[1,2-a]pyrazin-6-yl}-N-[4-(1H-pyrazol-4-yl)phenyl]benzamide; and
pharmaceutically acceptable salts thereof.
Also provided is at least one chemical entity chosen from
  • 3-{8-[(3-fluoro-4-methoxyphenyl)amino]imidazo[1,2-a]pyrazin-6-yl}-N-[4-(methylcarbamoyl)phenyl]benzamide;
  • N-[4-(methylcarbamoyl)phenyl]-3-(8-{[4-(piperazin-1-yl)phenyl]amino}imidazo[1,2-a]pyrazin-6-yl)benzamide;
  • 3-(8-{[4-(4-ethylpiperazin-1-yl)phenyl]amino}imidazo[1,2-a]pyrazin-6-yl)-N-[4-(methylcarbamoyl)phenyl]benzamide;
  • 4-{8-[(3,4-dimethoxyphenyl)amino]imidazo[1,2-a]pyrazin-6-yl}-N-[4-(methylcarbamoyl)phenyl]pyridine-2-carboxamide;
  • 3-{8-[(3,4-dimethoxyphenyl)amino]imidazo[1,2-a]pyrazin-6-yl}-5-methyl-N-[4-(methylcarbamoyl)phenyl]benzamide;
  • 4-(8-{[4-(1H-imidazol-2-yl)phenyl]amino}imidazo[1,2-a]pyrazin-6-yl)benzamide;
  • 5-{8-[(3,4-dimethoxyphenyl)amino]imidazo[1,2-a]pyrazin-6-yl}-N-[4-(methylcarbamoyl)phenyl]pyridine-3-carboxamide;
  • 3-{8-[(3,4-dimethoxyphenyl)amino]imidazo[1,2-a]pyrazin-6-yl}-5-fluoro-N-[4-(methylcarbamoyl)phenyl]benzamide;
  • N-[4-(methylcarbamoyl)phenyl]-3-(8-{[4-(3-oxopiperazin-1-yl)phenyl]amino}imidazo[1,2-a]pyrazin-6-yl)benzamide;
  • 5-{8-[(3,4-dimethoxyphenyl)amino]imidazo[1,2-a]pyrazin-6-yl}pyridine-3-carboxamide;
  • 4-{8-[(4-ethoxy-3-methoxyphenyl)amino]imidazo[1,2-a]pyrazin-6-yl}benzamide;
  • 4-{8-[(3,4,5-trimethoxyphenyl)amino]imidazo[1,2-a]pyrazin-6-yl}benzamide;
  • 3-{8-[(3,4-dimethoxyphenyl)amino]imidazo[1,2-a]pyrazin-6-yl}-5-fluorobenzamide;
  • 4-{8-[(3,4-dimethoxyphenyl)amino]imidazo[1,2-a]pyrazin-6-yl}-2-methylbenzamide;
  • 4-{8-[(3,4-dimethoxyphenyl)amino]imidazo[1,2-a]pyrazin-6-yl}-N,N-diethylbenzamide;
  • 6-[4-(1,4-diazepan-1-ylcarbonyl)phenyl]-N-(3,4-dimethoxyphenyl)imidazo[1,2-a]pyrazin-8-amine;
  • 4-{8-[(3,4-dimethoxyphenyl)amino]imidazo[1,2-a]pyrazin-6-yl}-2-fluorobenzamide;
  • 5-{8-[(3,4-dimethoxyphenyl)amino]imidazo[1,2-a]pyrazin-6-yl}-2-methylbenzamide;
  • 4-{8-[(3,4-dimethoxyphenyl)amino]imidazo[1,2-a]pyrazin-6-yl}-N-ethylbenzamide;
  • 2-chloro-4-{8-[(3,4-dimethoxyphenyl)amino]imidazo[1,2-a]pyrazin-6-yl}benzamide;
  • 3-{8-[(3,4-dimethoxyphenyl)amino]imidazo[1,2-a]pyrazin-6-yl}-5-methylbenzamide;
  • 4-(8-{[4-(propylcarbamoyl)phenyl]amino}imidazo[1,2-a]pyrazin-6-yl)benzamide;
  • 5-{8-[(4-ethoxy-3-methoxyphenyl)amino]imidazo[1,2-a]pyrazin-6-yl}-2-methyl-N-[4-(methylcarbamoyl)phenyl]benzamide;
  • 5-{8-[(3,4-dimethoxyphenyl)amino]imidazo[1,2-a]pyrazin-6-yl}-2-fluorobenzamide;
  • N-(3,4-dimethoxyphenyl)-6-[4-(piperazin-1-ylcarbonyl)phenyl]imidazo[1,2-a]pyrazin-8-amine;
  • 4-(8-{[4-(1H-imidazol-2-yl)phenyl]amino}imidazo[1,2-a]pyrazin-6-yl)benzamide;
  • 4-(8-{[3-methoxy-4-(propan-2-yloxy)phenyl]amino}imidazo[1,2-a]pyrazin-6-yl)benzamide;
  • 4-{8-[(3-methoxy-4-propoxyphenyl)amino]imidazo[1,2-a]pyrazin-6-yl}benzamide;
  • 3-{8-[(3,4-dimethoxyphenyl)amino]imidazo[1,2-a]pyrazin-6-yl}-4-fluorobenzamide;
  • 4-{8-[(3,4-dimethoxyphenyl)amino]imidazo[1,2-a]pyrazin-6-yl}-3-fluorobenzamide;
  • 4-{8-[(3,4-dimethoxyphenyl)amino]imidazo[1,2-a]pyrazin-6-yl}-3-methylbenzamide;
  • 4-(8-{[4-(2-hydroxyethoxy)-3-methoxyphenyl]amino}imidazo[1,2-a]pyrazin-6-yl)benzamide;
  • 4-{8-[(3-oxo-3,4-dihydro-2H-1,4-benzoxazin-6-yl)amino]imidazo[1,2-a]pyrazin-6-yl}benzamide;
  • 3-{8-[(3,4-dimethoxyphenyl)amino]imidazo[1,2-a]pyrazin-6-yl}-4-methylbenzamide;
  • 4-[8-({4-[2-(dimethylamino)ethoxy]-3-methoxyphenyl}amino)imidazo[1,2-a]pyrazin-6-yl]benzamide;
  • 4-{8-[(3,4-dimethoxyphenyl)amino]imidazo[1,2-a]pyrazin-6-yl}-N-(2-methoxyethyl)benzamide;
  • 4-{8-[(3,4-dimethoxyphenyl)amino]imidazo[1,2-a]pyrazin-6-yl}-N-(2-hydroxyethyl)benzamide;
  • 4-(8-{[3-methoxy-4-(2-methoxyethoxy)phenyl]amino}imidazo[1,2-a]pyrazin-6-yl)benzamide;
  • 4-{8-[(3,4-dimethoxyphenyl)amino]imidazo[1,2-a]pyrazin-6-yl}-N-[2-(methylamino)ethyl]benzamide;
  • 4-{8-[(3,4-dimethoxyphenyl)amino]imidazo[1,2-a]pyrazin-6-yl}-N-[2-(dimethylamino)ethyl]benzamide;
  • N-(3,4-dimethoxyphenyl)-6-[4-(morpholin-4-ylcarbonyl)phenyl]imidazo[1,2-a]pyrazin-8-amine;
  • 4-(8-{[4-(1H-imidazol-2-yl)-3-methylphenyl]amino}imidazo[1,2-a]pyrazin-6-yl)benzamide;
  • 4-{8-[(3,4-dimethoxyphenyl)amino]imidazo[1,2-a]pyrazin-6-yl}-2-methoxybenzamide;
  • 4-(8-{[3-methoxy-4-(piperidin-4-yloxy)phenyl]amino}imidazo[1,2-a]pyrazin-6-yl)benzamide;
  • 4-(8-{[4-(hydroxymethyl)phenyl]amino}imidazo[1,2-a]pyrazin-6-yl)benzamide;
  • 4-(8-{[4-(cyanomethoxy)-3-methoxyphenyl]amino}imidazo[1,2-a]pyrazin-6-yl)benzamide;
  • 4-(8-{[3-(1-hydroxyethyl)phenyl]amino}imidazo[1,2-a]pyrazin-6-yl)benzamide;
  • 4-(8-{[3-methoxy-4-(1H-pyrazol-5-yl)phenyl]amino}imidazo[1,2-a]pyrazin-6-yl)benzamide;
  • 5-{8-[(3,4-dimethoxyphenyl)amino]imidazo[1,2-a]pyrazin-6-yl}-2-methoxybenzamide;
  • 5-{8-[(3,4-dimethoxyphenyl)amino]imidazo[1,2-a]pyrazin-6-yl}pyridine-2-carboxamide;
  • 4-(8-{[4-(2-hydroxypropan-2-yl)phenyl]amino}imidazo[1,2-a]pyrazin-6-yl)benzamide;
  • and
  • 5-(8-{[4-(2-hydroxypropan-2-yl)phenyl]amino}imidazo[1,2-a]pyrazin-6-yl)-2-methyl-N-[4-(methylcarbamoyl)phenyl]benzamide, and
pharmaceutically acceptable salts thereof.
In all of the foregoing examples, the chemical entities can be administered alone, as mixtures, or in combination with other active agents.
Methods for obtaining the novel compounds described herein will be apparent to those of ordinary skill in the art, suitable procedures being described, for example, in the reaction schemes and examples below, and in the references cited herein.
Figure US08697699-20140415-C00009
Referring to Reaction Scheme 1, Step 1, a solution of a compound of Formula 101 in a polar solvent such as dichloromethane is added in excess (such as about 1.1 equivalents) to a compound of Formula 102, where R12 is a leaving group such as chloride. A tertiary amine such as N,N-diisopropylethylamine is added to the mixture in excess (such as about 3.3 equivalents). The mixture is stirred at room temperature for about 13 h. The product, a compound of Formula 103, is isolated and optionally purified.
Figure US08697699-20140415-C00010
Referring to Reaction Scheme 2, Step 2, a 1:1 ratio of a compound of Formula 103 and a compound of Formula 204 (where R13 is a halide such as Br) are taken up in an aqueous solution of base (such as 1M sodium carbonate) and an inert solvent such as 1,4-dioxane. The reaction mixture is sparged with nitrogen and stirred for 5 min. The resulting mixture is treated with about 0.2 equivalent of tetrakis(triphenylphosphine)palladium(0) and stirred to reflux for 2.5 h. The resulting product, a compound of Formula 205, is isolated and optionally purified.
Figure US08697699-20140415-C00011
Referring to Reaction Scheme 3, Step 1, a mixture of the salt of Formula 301 (such as about 0.7 equivalent), a compound of Formula 101 (such as about 1.0 equivalent), benzotriazol-1-yloxy-tris(dimethylamino)-phosphonium hexafluorophosphate (such as about 1.0 equivalent) and 4-methylmorpholine (such as about 3.3 equivalents) in an inert solvent such as DMF are stirred at room temperature for 18 h. The mixture is then stirred at 45° C. for 6 h. Then, methyl 4-amino-3-methylbenzoate (such as about 1.0 equivalent) is added and the resulting mixture is stirred at room temperature for an additional 18 h. Then, benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (such as about 0.7 equivalent) is added and the resulting reaction mixture is stirred at 45° C. for 18 h and then at ambient temperature for 6 d. The product, a compound of Formula 205, is isolated and optionally purified.
Figure US08697699-20140415-C00012
Referring to Reaction Scheme 4, Step 1, a compound of Formula 205 in an aqueous mixture of MeOH is treated with HCl (such as a 2N solution (pH<2)) and sonicated for approximately 45 min. Any solids that form are treated with a small volume of 2N HCl and Et2O. The resulting product, a salt of Formula 401, is optionally purified by trituration with acetonitrile.
Accordingly, provided is a method of treating a patient, for example, a mammal, such as a human, having a disease responsive to inhibition of Syk activity, comprising administrating to the patient having such a disease, an effective amount of at least one chemical entity described herein.
In some embodiments, the chemical entities described herein may also inhibit other kinases, such that disease, disease symptoms, and conditions associated with these kinases is also treated.
Methods of treatment also include inhibiting Syk activity and/or inhibiting B-cell activity, by inhibiting ATP binding or hydrolysis by Syk or by some other mechanism, in vivo, in a patient suffering from a disease responsive to inhibition of Syk activity, by administering an effective concentration of at least one chemical entity chosen described herein. An example of an effective concentration would be that concentration sufficient to inhibit Syk activity in vitro. An effective concentration may be ascertained experimentally, for example by assaying blood concentration of the chemical entity, or theoretically, by calculating bioavailability.
In some embodiments, the condition responsive to inhibition of Syk activity and/or B-cell activity is cancer, an allergic disorder and/or an autoimmune and/or inflammatory disease, and/or an acute inflammatory reaction.
Also provided is a method of treating a patient having cancer, an allergic disorder and/or an autoimmune and/or inflammatory disease, and/or an acute inflammatory reaction, by administering an effective amount of at least one chemical entity described herein.
In some embodiments, the conditions and diseases that can be affected using chemical entities described herein, include, but are not limited to: allergic disorders, including but not limited to eczema, allergic rhinitis or coryza, hay fever, bronchial asthma, urticaria (hives) and food allergies, and other atopic conditions; autoimmune and/or inflammatory diseases, including but not limited to psoriasis, Crohn's disease, irritable bowel syndrome, Sjogren's disease, tissue graft rejection, and hyperacute rejection of transplanted organs, asthma, systemic lupus erythematosus (and associated glomerulonephritis), dermatomyositis, multiple sclerosis, scleroderma, vasculitis (ANCA-associated and other vasculitides), autoimmune hemolytic and thrombocytopenic states, Goodpasture's syndrome (and associated glomerulonephritis and pulmonary hemorrhage), atherosclerosis, rheumatoid arthritis, chronic Idiopathic thrombocytopenic purpura (ITP), Addison's disease, Parkinson's disease, Alzheimer's disease, diabetes, septic shock, myasthenia gravis, and the like; acute inflammatory reactions, including but not limited to skin sunburn, inflammatory pelvic disease, inflammatory bowel disease, urethritis, uvitis, sinusitis, pneumonitis, encephalitis, meningitis, myocarditis, nephritis, osteomyelitis, myositis, hepatitis, gastritis, enteritis, dermatitis, gingivitis, appendicitis, pancreatitis, and cholocystitis; and cancer, including but not limited to, B-cell lymphoma, lymphoma (including Hodgkin's and non-Hodgkins lymphoma), hairy cell leukemia, multiple myeloma, chronic and acute myelogenous leukemia, and chronic and acute lymphocytic leukemia.
Syk is a known inhibitor of apoptosis in lymphoma B-cells. Defective apoptosis contributes to the pathogenesis and drug resistance of human leukemias and lymphomas. Thus, further provided is a method of promoting or inducing apoptosis in cells expressing Syk comprising contacting the cell with at least one chemical entity described herein.
Also provided are methods of treatment in which at least one chemical entity described herein is the only active agent given to a patient and also includes methods of treatment in which at least one chemical entity described herein is given to a patient in combination with one or more additional active agents.
Thus in some embodiments, a method of treating cancer, an allergic disorder and/or an autoimmune and/or inflammatory disease, and/or an acute inflammatory reaction comprises administering to a patient in need thereof an effective amount of at least one chemical entity described herein, together with a second active agent, which can be useful for treating a cancer, an allergic disorder and/or an autoimmune and/or inflammatory disease, and/or an acute inflammatory reaction. For example the second agent may be an anti-inflammatory agent. Treatment with the second active agent may be prior to, concomitant with, or following treatment with at least one chemical entity described herein. In some embodiments, at least one chemical entity described herein is combined with another active agent in a single dosage form. Suitable antitumor therapeutics that may be used in combination with at least one chemical entity described herein include, but are not limited to, chemotherapeutic agents, for example mitomycin C, carboplatin, taxol, cisplatin, paclitaxel, etoposide, doxorubicin, or a combination comprising at least one of the foregoing chemotherapeutic agents. Radiotherapeutic antitumor agents may also be used, alone or in combination with chemotherapeutic agents.
Chemical entities described herein can be useful as chemosensitizing agents, and, thus, can be useful in combination with other chemotherapeutic drugs, in particular, drugs that induce apoptosis.
A method for increasing sensitivity of cancer cells to chemotherapy, comprising administering to a patient undergoing chemotherapy a chemotherapeutic agent together with at least one chemical entity described herein in an amount sufficient to increase the sensitivity of cancer cells to the chemotherapeutic agent is also provided herein.
Examples of other chemotherapeutic drugs that can be used in combination with chemical entities described herein include topoisomerase I inhibitors (camptothesin or topotecan), topoisomerase II inhibitors (e.g. daunomycin and etoposide), alkylating agents (e.g. cyclophosphamide, melphalan and BCNU), tubulin directed agents (e.g. taxol and vinblastine), and biological agents (e.g. antibodies such as anti CD20 antibody, IDEC 8, immunotoxins, and cytokines).
In some embodiments, the chemical entities described herein are used in combination with Rituxan® (Rituximab) or other agents that work by selectively depleting CD20+ B-cells.
Included herein are methods of treatment in which at least one chemical entity described herein is administered in combination with an anti-inflammatory agent. Anti-inflammatory agents include but are not limited to NSAIDs, non-specific and COX-2 specific cyclooxgenase enzyme inhibitors, gold compounds, corticosteroids, methotrexate, tumor necrosis factor receptor (TNF) receptors antagonists, immunosuppressants and methotrexate.
Examples of NSAIDs include, but are not limited to ibuprofen, flurbiprofen, naproxen and naproxen sodium, diclofenac, combinations of diclofenac sodium and misoprostol, sulindac, oxaprozin, diflunisal, piroxicam, indomethacin, etodolac, fenoprofen calcium, ketoprofen, sodium nabumetone, sulfasalazine, tolmetin sodium, and hydroxychloroquine. Examples of NSAIDs also include COX-2 specific inhibitors (i.e., a compound that inhibits COX-2 with an IC50 that is at least 50-fold lower than the IC50 for COX-1) such as celecoxib, valdecoxib, lumiracoxib, etoricoxib and/or rofecoxib.
In a further embodiment, the anti-inflammatory agent is a salicylate. Salicylates include but are not limited to acetylsalicylic acid or aspirin, sodium salicylate, and choline and magnesium salicylates.
The anti-inflammatory agent may also be a corticosteroid. For example, the corticosteroid may be chosen from cortisone, dexamethasone, methylprednisolone, prednisolone, prednisolone sodium phosphate, and prednisone.
In some embodiments, the anti-inflammatory therapeutic agent is a gold compound such as gold sodium thiomalate or auranofin.
In some embodiments, the anti-inflammatory agent is a metabolic inhibitor such as a dihydrofolate reductase inhibitor, such as methotrexate or a dihydroorotate dehydrogenase inhibitor, such as leflunomide.
In some embodiments, combinations in which at least one anti-inflammatory compound is an anti-C5 monoclonal antibody (such as eculizumab or pexelizumab), a TNF antagonist, such as entanercept, or infliximab, which is an anti-TNF alpha monoclonal antibody are used.
In some embodiments, combinations in which at least one active agent is an immunosuppressant compound such as methotrexate, leflunomide, cyclosporine, tacrolimus, azathioprine, or mycophenolate mofetil are used.
Dosage levels of the order, for example, of from 0.1 mg to 140 mg per kilogram of body weight per day can be useful in the treatment of the above-indicated conditions (0.5 mg to 7 g per patient per day). The amount of active ingredient that may be combined with the vehicle to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. Dosage unit forms will generally contain from 1 mg to 500 mg of an active ingredient.
Frequency of dosage may also vary depending on the compound used and the particular disease treated. In some embodiments, for example, for the treatment of an allergic disorder and/or autoimmune and/or inflammatory disease, a dosage regimen of 4 times daily or less is used. In some embodiments, a dosage regimen of 1 or 2 times daily is used. It will be understood, however, that the specific dose level for any particular patient will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, and rate of excretion, drug combination and the severity of the particular disease in the patient undergoing therapy.
A labeled form of a chemical entity described herein can be used as a diagnostic for identifying and/or obtaining compounds that have the function of modulating an activity of a kinase as described herein. The chemical entities described herein may additionally be used for validating, optimizing, and standardizing bioassays.
By “labeled” herein is meant that the compound is either directly or indirectly labeled with a label which provides a detectable signal, e.g., radioisotope, fluorescent tag, enzyme, antibodies, particles such as magnetic particles, chemiluminescent tag, or specific binding molecules, etc. Specific binding molecules include pairs, such as biotin and streptavidin, digoxin and antidigoxin etc. For the specific binding members, the complementary member would normally be labeled with a molecule which provides for detection, in accordance with known procedures, as outlined above. The label can directly or indirectly provide a detectable signal.
EXAMPLES
The invention is further illustrated by the following non-limiting examples.
In the examples below, the following abbreviations have the following meanings. If an abbreviation is not defined, it has its generally accepted meaning.
DME = dimethyl ether
DMEM = Dulbecco's modified Eagle's medium
DMF = N,N-dimethylformamide
DMSO = dimethylsulfoxide
Et2O = diethylether
g = gram
h = hour
mg = milligram
min = minutes
mL = milliliter
mmol = millimoles
mM = millimolar
ng = nanogram
nm = nanometer
nM = nanomolar
PBS = phosphate buffered saline
μL = microliter
μM = micromolar
Example 1 Preparation of 4-(3-(8-(3,4-dimethoxyphenylamino)imidazo[1,2-a]pyrazin-6-yl)benzamido)benzoic acid (1)
Figure US08697699-20140415-C00013
Preparation of benzyl 4-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzamido)benzoate (2)
A mixture of benzyl 4-aminobenzoate (4.39 g, 19.3 mmol), 3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoyl chloride (4.30 g, 16.1 mmol) and N,N-diisopropylethylamine (7.49 g, 57.9 mmol) in methylene chloride (50 mL) was stirred at ambient temperature for 13 h. After this time, the reaction was concentrated under reduced pressure and purified by chromatography (silica, heptane to 1:3 ethyl acetate/heptane) to afford benzyl 4-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzamido)benzoate (3.07 g, 78% pure by mass, 27% yield) as a light yellow sticky foam that was used without further purification.
Preparation of benzyl 4-(3-(8-(3,4-dimethoxyphenylamino)imidazo[1,2-a]pyrazin-6-yl)benzamido)benzoate (3)
A mixture of 6-bromo-N-(3,4-dimethoxyphenyl)imidazo[1,2-a]pyrazin-8-amine (1.82 g, 5.22 mmol) and benzyl 4-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzamido)benzoate (3.06 g, 5.22 mmol, 78% by mass) in 1 M aqueous sodium carbonate (8 mL) and 1,4-dioxane (24 mL) was sparged with nitrogen with stirring for 5 min. The resulting mixture was treated with tetrakis(triphenylphosphine)palladium(0) (1.20 g, 1.04 mmol) and stirred at reflux for 2.5 h. After this time, the reaction was cooled to ambient temperature, diluted with ethyl acetate (100 mL) and washed with brine (100 mL). The aqueous layer was extracted with ethyl acetate (100 mL) and the combined organic layers were dried over sodium sulfate, filtered and the filtrate was concentrated under reduced pressure. The residue obtained was purified by chromatography (silica, methylene chloride to 1:4 ethyl acetate/methylene chloride) to afford benzyl 4-(3-(8-(3,4-dimethoxyphenylamino)imidazo[1,2-a]pyrazin-6-yl)benzamido)benzoate (2.02 g, 65%) as an off-white solid: mp 154-160° C.; 1H NMR (300 MHz, CDCl3) δ 10.72 (s, 1H), 9.57 (s, 1H), 8.74 (s, 1H), 8.57 (s, 1H), 8.25 (d, J=7.8 Hz, 1H), 8.08-7.94 (m, 7H), 7.69-7.60 (m, 3H), 7.50-7.34 (m, 5H), 6.92 (d, J=8.7 Hz, 1H), 5.36 (s, 2H), 3.78 (s, 3H), 3.73 (s, 3H); ESI MS m/z 600 [M+H]+; HPLC, 8.93 min, 95.9% (AUC).
Preparation of 4-(3-(8-(3,4-dimethoxyphenylamino)imidazo[1,2-a]pyrazin-6-yl)benzamido)benzoic acid (1)
A solution of benzyl 4-(3-(8-(3,4-dimethoxyphenylamino)imidazo[1,2-a]pyrazin-6-yl)benzamido)benzoate (1.75 g, 2.92 mmol) in ethyl acetate (500 mL) and ethanol (500 mL) at 60° C. was treated with 10% palladium on carbon (5.11 g, 50% water by weight) and sparged with hydrogen for 30 min at 60° C. then stirred under balloon pressure hydrogen at 60° C. for 30 min. After this time, the reaction was sparged with nitrogen for 10 min and filtered hot through diatomaceous earth. The filtrate was concentrated under reduced pressure and the residue obtained was exhaustively triturated: boiling acetonitrile (50 mL×2); then boiling methanol (50 mL); then boiling 1:1 methanol/acetonitrile (50 mL); then boiling acetonitrile (50 mL); then boiling 1:1 methanol/acetonitrile (50 mL) to afford 4-(3-(8-(3,4-dimethoxyphenylamino)imidazo[1,2-a]pyrazin-6-yl)benzamido)benzoic acid (458 mg, 31%) as an off-white solid: mp>250° C.; 1H NMR (300 MHz, DMSO-d6) δ 12.74 (br s, 1H), 10.67 (s, 1H), 9.57 (s, 1H), 8.74 (s, 1H), 8.56 (s, 1H), 8.25 (d, J=7.8 Hz, 1H), 8.80-7.89 (m, 7H), 7.69-7.53 (m, 3H), 6.93 (d, J=8.7 Hz, 1H), 3.78 (s, 3H), 3.71 (s, 3H); ESI MS m/z 510 [M+H]+; HPLC, 6.50 min, 95.6% (AUC).
Example 2 Preparation of 4-(3-(8-(3,4-dimethoxyphenylamino)imidazo[1,2-a]pyrazin-6-yl)benzamido)-3-methylbenzoic acid hydrochloride salt
Figure US08697699-20140415-C00014
Preparation of methyl 4-(3-(8-(3,4-dimethoxyphenylamino)imidazo[1,2-a]pyrazin-6-yl)benzamido)-3-methylbenzoate
A mixture of 3-(8-(3,4-dimethoxyphenylamino)imidazo[1,2-a]pyrazin-6-yl)benzoic acid hydrochloride salt (80 mg, 0.187 mmol), methyl 4-amino-3-methylbenzoate (46 mg, 0.281 mmol), benzotriazol-1-yloxy-tris(dimethylamino)-phosphonium hexafluorophosphate (124 mg, 0.281 mmol) and 4-methylmorpholine (95 mg, 0.937 mmol) in DMF (1.5 mL) was stirred at ambient temperature for 18 h. The mixture was then stirred at 45° C. for 6 h before additional methyl 4-amino-3-methylbenzoate (46 mg, 0.281 mmol) was added and the resulting mixture was stirred at ambient temperature for 18 h. To the reaction mixture was added (benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (97 mg, 0.187 mmol) and the resulting mixture was stirred at 45° C. for 18 h and then at ambient temperature for 6 d. After this time, the reaction was diluted with ethyl acetate (50 mL) and washed with water (30 mL) then 5% aqueous lithium chloride solution (30 mL). The organic layer was dried over sodium sulfate, filtered and the filtrate was concentrated under reduced pressure. The residue obtained was purified by chromatography (silica, methylene chloride to 39:1 methylene chloride/methanol) to afford methyl 4-(3-(8-(3,4-dimethoxyphenylamino)imidazo[1,2-a]pyrazin-6-yl)benzamido)-3-methylbenzoate (36 mg, 36%): ESI MS m/z 538 [M+H]+.
Preparation of 4-(3-(8-(3,4-dimethoxyphenylamino)imidazo[1,2-a]pyrazin-6-yl)benzamido)-3-methylbenzoic acid hydrochloride salt
A mixture of methyl 4-(3-(8-(3,4-dimethoxyphenylamino)imidazo[1,2-a]pyrazin-6-yl)benzamido)-3-methylbenzoate (36 mg, 0.070 mmol) in methanol (6 mL), THF (0.4 mL) and water (0.4 mL) was treated with lithium hydroxide (14 mg, 0.330 mmol) and the resulting mixture was stirred at ambient temperature for 20 h. After this time, the volatiles were removed under reduced pressure and the resulting solution was treated with 2 N hydrochloric acid (pH<2). The mixture was sonicated for approximately 45 min and the solids that formed were collected by filtration and washed with a small volume of 2 N hydrochloric acid and then with diethyl ether. The residue obtained was purified by trituration with acetonitrile to afford 4-(3-(8-(3,4-dimethoxyphenylamino)imidazo[1,2-a]pyrazin-6-yl)benzamido)-3-methylbenzoic acid hydrochloride salt (26 mg, 67%) as a yellow solid: mp 188-215° C.; 1H NMR (300 MHz, DMSO-d6) δ 10.12 (s, 1H), 9.83 (s, 1H), 8.77 (s, 1H), 8.59 (s, 1H), 8.31-8.24 (m, 1H), 8.10-7.99 (m, 3H), 7.88-7.83 (m, 3H), 7.70-7.45 (m, 3H), 6.96-6.93 (d, J=8.4 Hz, 1H), 3.79-3.77 (m, 6H), 2.34 (s, 3H); ESI MS m/z 524.9 [M+H]+; HPLC, 6.38 min, 95.4% (AUC)
Example 3
The following compounds were prepared using procedures similar to those described above. Those of ordinary skill in the art of organic synthesis will recognize when starting materials or reaction conditions should be varied to obtain the desired compound.
MS data reported in this example was obtained as follows:
MS conditions: Electrospray MS is performed on a MICROMASS LCT equipped with a LockSpray source for accurate mass measurements. Spectra are acquired in positive ion mode from 100-1000 Da at an acquisition rate of 1 spectrum/0.9 s with a 0.1 s interscan delay. The instrument is tuned for a resolution of 5000 (FWHM). Every 5th scan is taken from the reference position of the Lockspray source. Leucine enkephalin (556.2771 [M+H]+) is used as the reference or lock mass.
MH+
Structure Name m/z
Figure US08697699-20140415-C00015
 		3-{8-[(3-Methoxy-4- methylphenyl) amino]imidazo[1,2- a]pyrazin-6-yl}-N-[4-(1H- pyrazol-4- yl)phenyl]benzamide 516.9
Figure US08697699-20140415-C00016
 		3-{8-[(3,4- Dimethoxyphenyl)- amino]imidazo[1,2- a]pyrazin-6-yl}-N- (piperidin-4-yl)benzamide 473.3
Figure US08697699-20140415-C00017
 		N-(1H-1,3-Benzodiazol-6- yl)-3-{8-[(3,4- Dimethoxyphenyl)amino]- imidazo[1,2-a]pyrazin-6- yl}benzamide 506  
Figure US08697699-20140415-C00018
 		5-(3-(8-(3,4- Dimethoxyphenylamino)- imidazo[1,2-a]pyrazin-6- yl)benzamido)-N- methylpicolinamide 524.5
Figure US08697699-20140415-C00019
 		4-({3-[8-(1H-Indazol-6- ylamino)-imidazo[1,2- a]pyrazin-6- yl]benzene}amido)benzoic acid 490.2
Figure US08697699-20140415-C00020
 		3-{8-[(3,4- Dimethoxyphenyl)amino]- imidazo[1,2-a]pyrazin-6- yl}-N-(4- acetylphenyl)benzamide 508.2
Figure US08697699-20140415-C00021
 		6-[3-(2,3-Dihydro-1H- indol-1-ylcarbonyl) phenyl]-N-(3,4- Dimethoxyphenyl)- imidazo[1,2-a]pyrazin-8- amine 492.3
Figure US08697699-20140415-C00022
 		3-{8-[(3,4- Dimethoxyphenyl)amino]- imidazo[1,2-a]pyrazin-6- yl}-N-[4- (methylcarbamoyl)phenyl] benzamide 523.9
Figure US08697699-20140415-C00023
 		N-(4-Cyanophenyl)-3-{8- [(3,4- Dimethoxyphenyl)amino]- imidazo[1,2-a]pyrazin-6- yl}benzamide 491.7
Figure US08697699-20140415-C00024
 		4-[(3-{8-[(3,4- Dimethoxyphenyl)amino]- imidazo[1,2-a]pyrazin-6- yl}benzene)- amido]cyclohexe-1- carboxylic acid 516.1
Figure US08697699-20140415-C00025
 		4-{1-[(3-{8-[(3,4- Dimethoxyphenyl)- amino]imidazo[1,2- a]pyrazin-6-yl}- phenyl)formamido]-2- methylpropan-2-yl}benzoic acid 566.7
Figure US08697699-20140415-C00026
 		4-[(3-{8-[(3,4- Dimethoxyphenyl)- amino]imidazo[1,2- a]pyrazin-6- yl}benzene)amido]-2- methoxybenzoic acid 540.4
Figure US08697699-20140415-C00027
 		2-(Morpholin-4-yl)ethyl 4- [(3-{8-[(3,4- Dimethoxyphenyl)amino]- imidazo[1,2-a]pyrazin-6- yl}benzene)amido]benzoate 623.6
Figure US08697699-20140415-C00028
 		Ethyl 4-[(3-{8-[(3,4- Dimethoxyphenyl)- amino]imidazo[1,2- a]pyrazin-6- yl}benzene)amido]benzoate 538.9
Figure US08697699-20140415-C00029
 		2-{4-[(3-{8-[(3,4- Dimethoxyphenyl)- amino]imidazo[1,2- a]pyrazin-6-yl}- phenyl)carbonyl]piperazin- 1-yl}ethan-1-ol 503.5
Figure US08697699-20140415-C00030
 		4-[(3-{8-[(3,4- Dimethoxyphenyl)amino]- imidazo[1,2-a]pyrazin-6- yl}benzene)amido]-2- fluorobenzoic acid 528.3
Figure US08697699-20140415-C00031
 		3-{8-[(3,4- Dimethoxyphenyl)amino]- imidazo[1,2-a]pyrazin-6- yl}-N-[4- (methanesulfonylcarbamoyl)- phenyl]-benzamide 587.1
Figure US08697699-20140415-C00032
 		Benzyl 4-[(3-{8-[(3,4- Dimethoxyphenyl)- amino]imidazo[1,2- a]pyrazin-6-yl}- benzene)amido]benzoate 600.4
Figure US08697699-20140415-C00033
 		3-{8-[(3,4- Dimethoxyphenyl)amino]- imidazo[1,2-a]pyrazin-6- yl}-N-[4-(1H-pyrazol-4- yl)phenyl]benzamide 532.3
Figure US08697699-20140415-C00034
 		3-{8-[(3,4- Dimethoxyphenyl)amino]- imidazo[1,2-a]pyrazin-6- yl}-N-[4-(1H-1,2,3,4- tetrazol-5- yl)phenyl]benzamide 534.3
Figure US08697699-20140415-C00035
 		N-(1H-1,2,3-Benzotriazol- 5-yl)-3-{8-[(3,4- Dimethoxyphenyl)amino]- imidazo[1,2-a]pyrazin-6- yl}benzamide 507.3
Figure US08697699-20140415-C00036
 		N-(3,4-Dimethoxyphenyl)- 6-{3-[(4-methylpiperazin- 1-yl)carbonyl]phenyl}- imidazo[1,2-a]pyrazin-8- amine 473.4
Figure US08697699-20140415-C00037
 		4-[(3-{8-[(3,4- Dimethoxyphenyl)amino]- imidazo[1,2-a]pyrazin-6- yl}-2-methyl- benzene)amido]benzoic acid 524.4
Figure US08697699-20140415-C00038
 		3-[(4-{8-[(3,4- Dimethoxyphenyl)amino]- imidazo[1,2-a]pyrazin-6- yl}benzene)- amido]benzoic acid 510.8
Figure US08697699-20140415-C00039
 		N-(3,4-Dimethoxyphenyl)- 6-[3-(piperazin-1- ylcarbonyl)phenyl]- imidazo[1,2-a]pyrazin-8- amine 459.9
Figure US08697699-20140415-C00040
 		4-{[(3-{8-[(3,4- Dimethoxyphenyl)amino]- imidazo[1,2-a]pyrazin-6- yl}phenyl)- formamido]methyl}benzoic acid 524.8
Figure US08697699-20140415-C00041
 		1-[(3-{8-[(3,4- Dimethoxyphenyl)amino]- imidazo[1,2-a]pyrazin-6- yl}phenyl)- carbonyl]piperidine-4- carboxylic acid 502.5
Figure US08697699-20140415-C00042
 		N-{4- [(Dimethylamino)methyl]- phenyl}-3-(8-{[4- (morpholin-4- yl)phenyl]amino}- imidazo[1,2-a]pyrazin-6- yl)benzamide 548.6
Figure US08697699-20140415-C00043
 		3-{8-[(3,4- Dimethoxyphenyl)amino]- imidazo[1,2-a]pyrazin-6- yl}-N-[4-(piperazin-1- yl)phenyl]benzamide 550.2
Figure US08697699-20140415-C00044
 		3-{8-[(3,4- Dimethoxyphenyl)amino]- imidazo[1,2-a]pyrazin-6- yl}benzamide 390.7
Figure US08697699-20140415-C00045
 		3-(8-{[4-(Morpholin-4- yl)phenyl]- amino}imidazo[1,2- a]pyrazin-6-yl)-N-[4-(1H- pyrazol-4- yl)phenyl]benzamide 557.3
Figure US08697699-20140415-C00046
 		3-{8-[(3,4- Dimethoxyphenyl)amino]- imidazo[1,2-a]pyrazin-6- yl}-N,N- dimethylbenzamide 418.6
Figure US08697699-20140415-C00047
 		4-[(3-{8-[(3,4- Dimethoxyphenyl)amino]- imidazo[1,2-a]pyrazin-6- yl}benzene)-amido]-3- methylbenzoic acid 524.9
Figure US08697699-20140415-C00048
 		4-{8-[(3,4- Dimethoxyphenyl)amino]- imidazo[1,2-a]pyrazin-6- yl}benzamide 390.1
Figure US08697699-20140415-C00049
 		4-[(3-{8-[(3,4- Dimethoxyphenyl)amino]- imidazo[1,2-a]pyrazin-6- yl}benzene)- amido]benzoic acid 510.2
Figure US08697699-20140415-C00050
 		N-(3,4-Dimethoxyphenyl)- 6-[3-(1,2,3,4- tetrahydroisoquinolin-2- ylcarbonyl)- phenyl]imidazo[1,2- a]pyrazin-8-amine 506.7
Figure US08697699-20140415-C00051
 		3-{8-[(3,4- Dimethoxyphenyl)amino]- imidazo[1,2-a]pyrazin-6- yl}-N-[4-(1- hydroxyethyl)phenyl]- benzamide 510.7
Figure US08697699-20140415-C00052
 		3-{8-[(3,4- Dimethoxyphenyl)amino]- imidazo[1,2-a]pyrazin-6- yl}-N-(1-methylpiperidin- 4-yl)benzamide 487.5
Figure US08697699-20140415-C00053
 		3-{8-[(3,4- Dimethoxyphenyl)amino]- imidazo[1,2-a]pyrazin-6- yl}-N-[3- (methylsulfamoyl)phenyl]- benzamide 559.3
Figure US08697699-20140415-C00054
 		4-[(3-{8-[(3,4- Dimethoxyphenyl)amino]- imidazo[1,2-a]pyrazin-6- yl}benzene)- (methyl)amido]benzoic acid 524.5
Figure US08697699-20140415-C00055
 		3-{8-[(3,4- Dimethoxyphenyl)amino]- imidazo[1,2-a]pyrazin-6- yl}-N-phenylbenzamide 466.6
Figure US08697699-20140415-C00056
 		3-{8-[(3,4- Dimethoxyphenyl)amino]- imidazo[1,2-a]pyrazin-6- yl}-N-(1H-indazol-5- yl)benzamide 506.2
Figure US08697699-20140415-C00057
 		4-[(4-{8-[(3,4- Dimethoxyphenyl)amino]- imidazo[1,2-a]pyrazin-6- yl}benzene)- amido]benzoic acid 510.9
Figure US08697699-20140415-C00058
 		3-{8-[(3,4- Dimethoxyphenyl)amino]- imidazo[1,2-a]pyrazin-6- yl}-N-[4-(1H-imidazol-2- yl)phenyl]benzamide 532.6
Figure US08697699-20140415-C00059
 		2-{4-[(3-{8-[(3,4- Dimethoxyphenyl)- amino]imidazo[1,2- a]pyrazin-6- yl}benzene)amido]phenyl} acetic acid 524.6
Figure US08697699-20140415-C00060
 		4-[(3-{8-[(3,4- Dimethoxyphenyl)amino]- imidazo[1,2-a]pyrazin-6- yl}phenyl)- carbonyl]piperazin-2-one 473.3
Figure US08697699-20140415-C00061
 		3-{8-[(3,4- Dimethoxyphenyl)amino]- imidazo[1,2-a]pyrazin-6- yl}-N-[4- (methylsulfamoyl)phenyl]- benzamide 559.3
Figure US08697699-20140415-C00062
 		4-{8-[(3,4- Dimethoxyphenyl)amino]- imidazo[1,2-a]pyrazin-6- yl}-N-[2-(pyridin-4- yl)ethyl]benzamide 495.3
Figure US08697699-20140415-C00063
 		3-{8-[(3,4- Dimethoxyphenyl)amino]- imidazo[1,2-a]pyrazin-6- yl}-N-methylbenzamide 404.8
Figure US08697699-20140415-C00064
 		3-{8-[(3,4- Dimethoxyphenyl)amino] imidazo[1,2-a]pyrazin-6- yl}-N-(pyridin-4- ylmethyl)-benzamide 481.2
Figure US08697699-20140415-C00065
 		3-[8-({4-[(4- ethylpiperazin-1- yl)methyl]- phenyl}amino)imidazo[1,2- a]pyrazin-6-yl]-N-[4-(1H- pyrazol-4-yl)phenyl]- benzamide 598.5
Figure US08697699-20140415-C00066
 		N-{4-[(1R)-1- Aminoethyl]phenyl}-3-{8- [(3,4- dimethoxyphenyl)amino]- imidazo[1,2-a]pyrazin-6- yl}benzamide 492.3
Figure US08697699-20140415-C00067
 		6-[3-(1,4-Diazepan-1- ylcarbonyl)-phenyl]-N- (3,4-dimethoxyphenyl)- imidazo[1,2-a]pyrazin-8- amine 473.7
Figure US08697699-20140415-C00068
 		3-{8-[(3,4- Dimethoxyphenyl)amino]- imidazo[1,2-a]pyrazin-6- yl}-N-[4-(1H-pyrazol-5- yl)phenyl]benzamide 532.2
Figure US08697699-20140415-C00069
 		5-[(3-{8-[(3,4- Dimethoxyphenyl)amino]- imidazo[1,2-a]pyrazin-6- yl}benzene)- amido]pyridine-2- carboxylic acid 511.9
Figure US08697699-20140415-C00070
 		3-{8-[(3,4- Dimethoxyphenyl)amino]- imidazo[1,2-a]pyrazin-6- yl}-N-[(6-hydroxypyridin- 3-yl)methyl]benzamide 497.9
Figure US08697699-20140415-C00071
 		3-{8-[(3,4- Dimethoxyphenyl)amino]- imidazo[1,2-a]pyrazin-6- yl}-N-{4- [(methylcarbamoyl)methyl]- phenyl} benzamide 537.5
Figure US08697699-20140415-C00072
 		N-(4-Carbamoylphenyl)-3- {8-[(3,4- dimethoxyphenyl)amino]- imidazo[1,2-a]pyrazin-6- yl}benzamide 509.4
Figure US08697699-20140415-C00073
 		4-[(3-{8-[(3,4- Dimethoxyphenyl)amino]- imidazo[1,2-a]pyrazin-6- yl}benzene)- sulfonamido]benzoic acid 544.6
Figure US08697699-20140415-C00074
 		3-{8-[(3,4- Dimethoxyphenyl)amino]- imidazo[1,2-a]pyrazin-6- yl}-N-(4-fluorophenyl) benzamide 484.7
Figure US08697699-20140415-C00075
 		methyl 4-[(3-{8-[(3,4- Dimethoxyphenyl)- amino]imidazo[1,2- a]pyrazin-6- yl}benzene)amido]benzoate 524.6
Figure US08697699-20140415-C00076
 		N-{4-[(1S)-1- Aminoethyl]phenyl}-3-{8- [(3,4- dimethoxyphenyl)amino]- imidazo[1,2-a]pyrazin-6- yl}benzamide 492.5 (M + H—NH3)
Figure US08697699-20140415-C00077
 		3-{8-[(3,4- Dimethoxyphenyl)amino]- imidazo[1,2-a]pyrazin-6- yl}-N-(4- methylphenyl)benzamide 480.4
Figure US08697699-20140415-C00078
 		2-{4-[(3-{8-[(3,4- Dimethoxyphenyl)- amino]imidazo[1,2- a]pyrazin-6-yl}- phenyl)carbonyl]piperazin- 1-yl}acetic acid 517.6
Figure US08697699-20140415-C00079
 		3-{8-[(3,4- Dimethoxyphenyl)amino]- imidazo[1,2-a]pyrazin-6- yl}-N-(6-hydroxypyridin- 3-yl)benzamide 483.8
Figure US08697699-20140415-C00080
 		1-[(3-{8-[(3,4- Dimethoxyphenyl)amino]- imidazo[1,2-a]pyrazin-6- yl}phenyl)- carbonyl]piperidin-4-ol 474.3
Figure US08697699-20140415-C00081
 		3-{8-[(3,4- Dimethoxyphenyl)amino]- imidazo[1,2-a]pyrazin-6- yl}-N-(1H-indazol-6-yl)- benzamide 506.0
Figure US08697699-20140415-C00082
 		3-[(3-{8-[(3,4- Dimethoxyphenyl)amino]- imidazo[1,2-a]pyrazin-6- yl}benzene)- amido]benzoic acid 510.8
Figure US08697699-20140415-C00083
 		4-{8-[(3,4- Dimethoxyphenyl)amino]- imidazo[1,2-a]pyrazin-6- yl}-N-(pyridin-3-ylmethyl) benzamide 481.1
Figure US08697699-20140415-C00084
 		4-[(3-{8-[(3,4- Dimethoxyphenyl)amino]- imidazo[1,2-a]pyrazim-6- yl}benzene)- amido]-2-methylbenzoic acid 524.6
Figure US08697699-20140415-C00085
 		N-{4- [(methylamino)methyl]- phenyl}-3-(8-{[4-(morpholin- 4-yl)phenyl]amino}- imidazo[1,2-a]pyrazin-6- yl)benzamide 534.1
Figure US08697699-20140415-C00086
 		4-{[3-(8-{[4-(morpholin-4- yl)phenyl]- amino}imidazo[1,2- a]pyrazin-6- yl)benzene]amido}benzoic acid 535.2
Figure US08697699-20140415-C00087
 		3-[8-({4-[2- (dimethylamino)ethoxy]-3- methoxyphenyl}amino)- imidazo[1,2-a]pyrazin-6-yl]- N-[4-(1H-pyrazol-4-yl)- phenyl]benzamide 589.7
Figure US08697699-20140415-C00088
 		N-[4- (hydroxycarbamoyl)phenyl]- 3-(8-{[4-(morpholin-4- yl)phenyl]amino}- imidazo[1,2-a]pyrazin-6- yl)benzamide 548.2
Figure US08697699-20140415-C00089
 		3-(8-{[4-(morpholin-4- yl)phenyl]amino}- imidazo[1,2-a]pyrazin-6- yl)-N-[4-(1H-1,2,4-triazol- 3-yl)phenyl]benzamide 558.3
Figure US08697699-20140415-C00090
 		3-(8-{[3-methoxy-4- (morpholin-4-yl)- phenyl]amino}imidazo[1,2- a]pyrazin-6-yl)-N-[4-(1H- pyrazol-4-yl)phenyl]- benzamide 587.2
Figure US08697699-20140415-C00091
 		3-(8-{[3-methoxy-4- (morpholin-4- ylcarbonyl)phenyl]amino}- imidazo[1,2-a]pyrazin-6- yl)-N-[4-(1H-pyrazol-4- yl)phenyl]benzamide 615.7
Figure US08697699-20140415-C00092
 		N-(1H-1,3-benzodiazol-5- yl)-3-(8-{[4-(morpholin-4- yl)phenyl]amino}- imidazo[1,2-a]pyrazin-6- yl)benzamide 531.4
Figure US08697699-20140415-C00093
 		3-[8-({4-[2- (dimethylamino)ethoxy]-3- methoxyphenyl}amino)- imidazo[1,2-a]pyrazin-6-yl]- N-[4-(1H-pyrazol-4- yl)phenyl]benzamide 589.7
Figure US08697699-20140415-C00094
 		N-[4- (hydroxycarbamoyl)phenyl]- 3-(8-{[4-(morpholin- 4yl)phenyl]amino}- imidazo[1,2-a]pyrazin-6- yl)benzamide 548.2
Figure US08697699-20140415-C00095
 		3-(8-{[4-(morpholin-4- yl)phenyl]amino}- imidazo[1,2-a]pyrazin-6- yl)-N-[4-(1H-1,2,4-triazol- 3-yl)phenyl]benzamide 558.3
Figure US08697699-20140415-C00096
 		3-(8-{[3-methoxy-4- (morpholin-4- yl)phenyl]amino}imidazo- [1,2-a]pyrazin-6-yl)-N-[4- (1H-pyrazol-4-yl)phenyl]- benzamide 587.2
Figure US08697699-20140415-C00097
 		3-(8-{[3-methoxy-4- (morpholin-4- ylcarbonyl)phenyl]amino}- imidazo[1,2-a]pyrazin-6- yl)-N-[4-(1H-pyrazol-4- yl)phenyl]benzamide 615.7
Figure US08697699-20140415-C00098
 		N-(1H-1,3-benzodiazol-5- yl)-3-(8-{[4-(morpholin-4- yl)phenyl]amino}- imidazo[1,2-a]pyrazin-6- yl)benzamide 531.4
Figure US08697699-20140415-C00099
 		5-{8-[(3,4- dimethoxyphenyl)amino]- imidazo[1,2-a]pyrazin-6- yl}-N-[4-(1H-pyrazol-4- yl)phenyl]pyridine-3- carboxamide 533.2
Figure US08697699-20140415-C00100
 		N-[4-(1H-imidazol-4- yl)phenyl]-3-(8-{[4- (morpholin-4- yl)phenyl]amino}- imidazo[1,2-a]pyrazin-6- yl)benzamide 557.2
Figure US08697699-20140415-C00101
 		3-{8-[(3,4- diethoxyphenyl)amino]- imidazo[1,2-a]pyrazin-6- yl}-N-[4-(1H-pyrazol-4- yl)phenyl]benzamide 560.4
Figure US08697699-20140415-C00102
 		3-{8-[(3,4- dimethoxyphenyl)amino]- imidazo[1,2-a]pyrazin-6- yl}-N-[3-methyl-4-(1H- pyrazol-4- yl)phenyl]benzamide 546.4
Figure US08697699-20140415-C00103
 		3-{8-[(3-ethoxy-4- methoxyphenyl)- amino]imidazo[1,2- a]pyrazin-6-yl}-N-[4- (methylcarbamoyl)phenyl] benzamide 537.2
Figure US08697699-20140415-C00104
 		3-{8-[(4-ethoxy-3- methoxyphenyl)- amino]imidazo[1,2- a]pyrazin-6-yl}-N-[4- (methylcarbamoyl)phenyl] benzamide 537.2
Figure US08697699-20140415-C00105
 		3-{8-[(4-methoxy-3- methylphenyl)- amino]imidazo[1,2- a]pyrazin-6-yl}-N-[4- (methylcarbamoyl)phenyl] benzamide 507.3
Figure US08697699-20140415-C00106
 		3-{8-[(6- methoxypyridazin-3- yl)amino]-imidazo[1,2- a]pyrazin-6-yl}-N-[4- (methylcarbamoyl)phenyl] benzamide 495.6
Figure US08697699-20140415-C00107
 		3-{8-[(3,4- dimethoxyphenyl)amino]- imidazo[1,2-a]pyrazin-6- yl}-N-[2-methyl-4-(1H- pyrazol-4- yl)phenyl]benzamide 546.4
Figure US08697699-20140415-C00108
 		3-{8-[(1-ethyl-1H-pyrazol- 4-yl)amino]-imidazo[1,2- a]pyrazin-6-yl}-N-[4-(1H- pyrazol-4- yl)phenyl]benzamide 490.3
Figure US08697699-20140415-C00109
 		3-{8-[(3-fluoro-4- methoxyphenyl)amino] imidazo[1,2-a]pyrazin-6-yl}- N-[4- (methylcarbamoyl)phenyl] benzamide 511.4
Figure US08697699-20140415-C00110
 		N-[4- (methylcarbamoyl)phenyl]- 3-(8-{[4-(piperazin-1- yl)phenyl]amino}imidazo [1,2-a]pyrazin-6- yl)benzamide 547.4
Figure US08697699-20140415-C00111
 		3-(8-{[4-(4-ethylpiperazin-1- yl)phenyl]amino}imidazo- [1,2-a]pyrazin-6-yl)-N-[4- (methylcarbamoyl)phenyl] benzamide 575.7
Figure US08697699-20140415-C00112
 		4-{8-[(3,4- dimethoxyphenyl)amino] imidazo[1,2-a]pyrazin-6- yl}-N-[4- (methylcarbamoyl)phenyl] pyridine-2-carboxamide 524.8
Figure US08697699-20140415-C00113
 		3-{8-[(3,4- dimethoxyphenyl)amino]- imidazo[1,2-a]pyrazin-6- yl}-5-methyl-N-[4- (methylcarbamoyl)phenyl] benzamide 537.7
Figure US08697699-20140415-C00114
 		4-(8-{[4-(1H-imidazol-2- yl)phenyl]amino}imidazo- [1,2-a]pyrazin-6- yl)benzamide 537.3
Figure US08697699-20140415-C00115
 		5-{8-[(3,4- dimethoxyphenyl)amino]- imidazo[1,2-a]pyrazin-6- yl}-N-[4- (methylcarbamoyl)phenyl] pyridine-3-carboxamide 524.7
Figure US08697699-20140415-C00116
 		3-{8-[(3,4- dimethoxyphenyl)amino]- imidazo[1,2-a]pyrazin-6- yl}-5-fluoro-N-[4- (methylcarbamoyl)phenyl] benzamide 541.1
Figure US08697699-20140415-C00117
 		N-[4- (methylcarbamoyl)phenyl]- 3-(8-{[4-(3-oxopiperazin-1- yl)phenyl]amino}imidazo- [1,2-a]pyrazin-6- yl)benzamide 561.3
Figure US08697699-20140415-C00118
 		5-{8-[(3,4- dimethoxyphenyl)amino]- imidazo[1,2-a]pyrazin-6- yl}pyridine-3-carboxamide 391.3
Figure US08697699-20140415-C00119
 		4-{8-[(4-ethoxy-3- methoxyphenyl)amino]- imidazo[1,2-a]pyrazin-6- yl}benzamide 404.3
Figure US08697699-20140415-C00120
 		4-{8-[(3,4,5- trimethoxyphenyl)amino]- imidazo[1,2-a]pyrazin-6- yl}benzamide 420.2
Figure US08697699-20140415-C00121
 		3-{8-[(3,4- dimethoxyphenyl)amino]- imidazo[1,2-a]pyrazin-6- yl}-5-fluorobenzamide 408.1
Figure US08697699-20140415-C00122
 		4-{8-[(3,4- dimethoxyphenyl)amino]- imidazo[1,2-a]pyrazin-6- yl}-2-methylbenzamide 404.7
Figure US08697699-20140415-C00123
 		4-{8-[(3,4- dimethoxyphenyl)amino]- imidazo[1,2-a]pyrazin-6- yl}-N,N-diethylbenzamide 446.4
Figure US08697699-20140415-C00124
 		6-[4-(1,4-diazepan-1- ylcarbonyl)phenyl]-N-(3,4- dimethoxyphenyl)imidazo- [1,2-a]pyrazin-8-amine 473.1
Figure US08697699-20140415-C00125
 		4-{8-[(3,4- dimethoxyphenyl)amino]- imidazo[1,2-a]pyrazin-6- yl}-2-fluorobenzamide 408.3
Figure US08697699-20140415-C00126
 		5-{8-[(3,4- dimethoxyphenyl)amino]- imidazo[1,2-a]pyrazin-6- yl}-2-methylbenzamide 404.1
Figure US08697699-20140415-C00127
 		4-{8-[(3,4- dimethoxyphenyl)amino]- imidazo[1,2-a]pyrazin-6- yl}-N-ethylbenzamide 418.6
Figure US08697699-20140415-C00128
 		2-chloro-4-{8-[(3,4- dimethoxyphenyl)amino]- imidazo[1,2-a]pyrazin-6- yl}benzamide 424.1
Figure US08697699-20140415-C00129
 		3-{8-[(3,4- dimethoxyphenyl)amino]- imidazo[1,2-a]pyrazin-6- yl}-5-methylbenzamide 404.6
Figure US08697699-20140415-C00130
 		4-(8-{[4- (propylcarbamoyl)phenyl]- amino}imidazo[1,2- a]pyrazin-6-yl)benzamide 415.6
Figure US08697699-20140415-C00131
 		5-{8-[(4-ethoxy-3- methoxyphenyl)amino]- imidazo[1,2-a]pyrazin-6-yl}- 2-methyl-N-[4- (methylcarbamoyl)phenyl] benzamide 551.8
Figure US08697699-20140415-C00132
 		5-{8-[(3,4- dimethoxyphenyl)amino]- imidazo[1,2-a]pyrazin-6- yl}-2-fluorobenzamide 408.7
Figure US08697699-20140415-C00133
 		N-(3,4-dimethoxyphenyl)- 6-[4-(piperazin-1- ylcarbonyl)phenyl]imidazo- [1,2-a]pyrazin-8-amine 459.4
Figure US08697699-20140415-C00134
 		4-(8-{[4-(1H-imidazol-2- yl)phenyl]amino}imidazo- [1,2-a]pyrazin-6- yl)benzamide 396.2
Figure US08697699-20140415-C00135
 		4-(8-{[3-methoxy-4- (propan-2- yloxy)phenyl]amino}- imidazo[1,2-a]pyrazin-6- yl)benzamide 418.2
Figure US08697699-20140415-C00136
 		4-{8-[(3-methoxy-4- propoxyphenyl)amino]- imidazo[1,2-a]pyrazin-6- yl}benzamide 418.2
Figure US08697699-20140415-C00137
 		3-{8-[(3,4- dimethoxyphenyl)amino]- imidazo[1,2-a]pyrazin-6- yl}-4-fluorobenzamide 408.1
Figure US08697699-20140415-C00138
 		4-{8-[(3,4- dimethoxyphenyl)amino]- imidazo[1,2-a]pyrazin-6- yl}-3-fluorobenzamide 408.1
Figure US08697699-20140415-C00139
 		4-{8-[(3,4- dimethoxyphenyl)amino]- imidazo[1,2-a]pyrazin-6- yl}-3-methylbenzamide 404.1
Figure US08697699-20140415-C00140
 		4-(8-{[4-(2- hydroxyethoxy)-3- methoxyphenyl]amino}- imidazo[1,2-a]pyrazin-6- yl)benzamide 420.1
Figure US08697699-20140415-C00141
 		4-{8-[(3-oxo-3,4-dihydro- 2H-1,4-benzoxazin-6- yl)amino]imidazo[1,2- a]pyrazin-6-yl}benzamide 400.6
Figure US08697699-20140415-C00142
 		3-{8-[(3,4- dimethoxyphenyl)amino]- imidazo[1,2-a]pyrazin-6- yl}-4-methylbenzamide 404.7
Figure US08697699-20140415-C00143
 		4-[8-({4-[2- (dimethylamino)ethoxy]-3- methoxyphenyl}amino)- imidazo[1,2-a]pyrazin-6- yl]benzamide 447.3
Figure US08697699-20140415-C00144
 		4-{8-[(3,4- dimethoxyphenyl)amino]- imidazo[1,2-a]pyrazin-6- yl}-N-(2- methoxyethyl)benzamide 448.9
Figure US08697699-20140415-C00145
 		4-{8-[(3,4- dimethoxyphenyl)amino]- imidazo[1,2-a]pyrazin-6- yl}-N-(2- hydroxyethyl)benzamide 434.3
Figure US08697699-20140415-C00146
 		4-(8-{[3-methoxy-4-(2- methoxyethoxy)phenyl]- amino}imidazo[1,2-a]- pyrazin-6-yl)benzamide 434.4
Figure US08697699-20140415-C00147
 		4-{8-[(3,4- dimethoxyphenyl)amino]- imidazo[1,2-a]pyrazin-6- yl}-N-[2- (methylamino)ethyl] benzamide 447.3
Figure US08697699-20140415-C00148
 		4-{8-[(3,4- dimethoxyphenyl)amino] imidazo[1,2-a]pyrazin-6- yl}-N-[2- (dimethylamino)ethyl] benzamide 461.4
Figure US08697699-20140415-C00149
 		N-(3,4-dimethoxyphenyl)- 6-[4-(morpholin-4- ylcarbonyl)phenyl]imidazo- [1,2-a]pyrazin-8-amine 460.3
Figure US08697699-20140415-C00150
 		4-(8-{[4-(1H-imidazol-2- yl)-3- methylphenyl]amino}- imidazo[1,2-a]pyrazin-6- yl)benzamide 410.2
Figure US08697699-20140415-C00151
 		4-{8-[(3,4- dimethoxyphenyl)amino]- imidazo[1,2-a]pyrazin-6- yl}-2-methoxybenzamide 420.3
Figure US08697699-20140415-C00152
 		4-(8-{[3-methoxy-4- (piperidin-4- yloxy)phenyl]amino}- imidazo[1,2-a]pyrazin-6- yl)benzamide 459.2
Figure US08697699-20140415-C00153
 		4-(8-{[4- (hydroxymethyl)phenyl]- amino}imidazo[1,2-a]pyrazin- 6-yl)benzamide 342.4
Figure US08697699-20140415-C00154
 		4-(8-{[4-(cyanomethoxy)- 3-methoxyphenyl]amino}- imidazo[1,2-a]pyrazin-6- yl)benzamide 415.2
Figure US08697699-20140415-C00155
 		4-(8-{[3-(1- hydroxyethyl)phenyl]amino}- imidazo[1,2-a]pyrazin-6- yl)benzamide 374.3
Figure US08697699-20140415-C00156
 		4-(8-{[3-methoxy-4-(1H- pyrazol-5- yl)phenyl]amino}imidazo- [1,2-a]pyrazin-6- yl)benzamide 426.2
Figure US08697699-20140415-C00157
 		5-{8-[(3,4- dimethoxyphenyl)amino]- imidazo[1,2-a]pyrazin-6- yl}-2-methoxybenzamide 420.2
Figure US08697699-20140415-C00158
 		5-{8-[(3,4- dimethoxyphenyl)amino]- imidazo[1,2-a]pyrazin-6- yl}pyridine-2-carboxamide 391.7
Figure US08697699-20140415-C00159
 		4-(8-{[4-(2- hydroxypropan-2- yl)phenyl]amino}imidazo- [1,2-a]pyrazin-6- yl)benzamide 388.6
Figure US08697699-20140415-C00160
 		5-(8-{[4-(2- hydroxypropan-2- yl)phenyl]amino}imidazo- [1,2-a]pyrazin-6-yl)-2- methyl-N-[4- (methylcarbamoyl)phenyl] benzamide 517.6
Example 4 Biochemical Syk Assay
A generalized procedure for one standard biochemical Syk Kinase Assay that can be used to test compounds disclosed in this application is as follows.
A master mix minus Syk enzyme is prepared containing 1× Cell Signaling kinase buffer (25 mM Tris-HCl, pH 7.5, 5 mM beta-glycerophosphate, 2 mM dithiothreitol, 0.1 mM Na3VO4, 10 mM MgCl2), 0.5 μM Promega PTK Biotinylated peptide substrate 1, 0.01% casein, 0.01% Triton-X100, and 0.25% glycerol. A master mix plus Syk enzyme is prepared containing 1× Cell Signaling kinase buffer, 0.5 μM PTK Biotinylated peptide substrate 1, 0.01% casein, 0.01% Triton-X100, 0.25% glycerol and 0.4 ng/well Syk enzyme. Syk enzyme is purchased from Cell Signaling Technologies, expressed in baculovirus and is an N-terminally GST-tagged full length human wildtype Syk. The Syk nucleotide sequence accession number is NM—003177, and accession number for the encoded Syk enzyme is NP—003168. The Syk protein was purified in one step using glutathione-agarose. The purity of the final protein preparation was assessed by SDS-PAGE and Coomassie staining. A solution of 200 μM ATP is prepared in water and adjusted to pH7.4 with 1N NaOH. A quantity of 1.25 μL of compound in 5% DMSO is transferred to a 96-well ½ area Costar polystyrene plate. Compounds are tested singly and with an 11-point dose-responsive curve (starting concentration is 10-1 μM; 1:2 dilution). A quantity of 18.75 μL of master mix minus enzyme (as a negative control) and master mix plus enzyme is transferred to appropriate wells in 96-well ½ area costar polystyrene plate. 5 μL of 200 μM ATP is added to that mixture in the 96-well ½ area Costar polystyrene plate for final ATP concentration of 40 μM. The reaction is allowed to incubate for 1 hour at room temperature. The reaction is stopped with Perkin Elmer 1× detection buffer containing 30 mM EDTA, 80 nM SA-APC, and 4 nM PT66 Ab. The plate is read using time-resolved fluorescence with a Perkin Elmer Envision using excitation filter 330 nm, emission filter 665 nm, and 2nd emission filter 615 nm. IC50 values are subsequently calculated using a log-logit regression algorithm.
Example 5 Ramos Cell pBLNK(Y96) Assay
Another generalized procedure for a standard cellular Syk Kinase Assay that can be used to test compounds disclosed in this application is as follows.
Ramos cells are serum starved at 2×106 cells/ml in serum-free RPMI for 1 hour in an upright T175 Falcon TC flask. Cells are centrifuged (1100 rpm×5 mM) and incubated at a density of 0.5×107 cells/ml in the presence of test compound or DMSO controls for 1 hr at 37° C. Cells are then stimulated by incubating with 10 μg/ml anti-human IgM F(ab)2 for 5 minutes at 37° C. Cells are pelleted, lysed in 40 ul cell lysis buffer, and mixed with Invitrogen SDS-PAGE loading buffer. 20 ul of cell lysate for each sample are subject to SDS-PAGE and western blotting with anti-phosphoBLNK(Tyr96) antibody (Cell Signaling Technology #3601) to assess Syk activity and anti-Syk antibody (BD Transduction Labs #611116) to control for total protein load in each lysate. The images are detected using fluorescent secondary detection systems and the LiCor Odyssey instrument.
Example 6 B-Cell Proliferation Assay
A generalized procedure for a standard cellular B-cell proliferation assay that can be used to test compounds disclosed in this application is as follows.
B-cells are purified from spleens of 8-16 week old Balb/c mice using a B-cell isolation kit (Miltenyi Biotech, Cat #130-090-862). Test compounds are diluted in 0.25% DMSO and incubated with 2.5×105 purified mouse splenic B-cells for 30 min prior to addition of 10 μg/ml of an anti-mouse IgM antibody (Southern Biotechnology Associates Cat #1022-01) in a final volume of 100 μl. Following 24 hr incubation, 1 μCi 3H-thymidine is added and plates are incubated an additional 36 hr prior to harvest using the manufacturer's protocol for SPA[3H] thymidine uptake assay system (Amersham Biosciences # RPNQ 0130). SPA-bead based fluorescence is counted in a microbeta counter (Wallace Triplex 1450, Perkin Elmer).
Example 7 T Cell Proliferation Assay
A generalized procedure for a standard T cell proliferation assay that can be used to test compounds disclosed in this application is as follows.
T cells are purified from spleens of 8-16 week old Balb/c mice using a Pan T cell isolation kit (Miltenyi Biotech, Cat #130-090-861). Test compounds are diluted in 0.25% DMSO and incubated with 2.5×105 purified mouse splenic T cells in a final volume of 100 μl in flat clear bottom plates precoated for 90 min at 37° C. with 10 μg/ml each of anti-CD3 (BD #553057) and anti-CD28 (BD #553294) antibodies. Following 24 hr incubation, 1 μCi 3H-thymidine is added and plates incubated an additional 36 hr prior to harvest using the manufacturer's protocol for SPA[3H] thymidine uptake assay system (Amersham Biosciences # RPNQ 0130). SPA-bead based fluorescence was counted in a microbeta counter (Wallace Triplex 1450, Perkin Elmer).
Example 8 CD69 Inhibition Assay
A generalized procedure for a standard assay for the inhibition of B cell activity that can be used to test compounds disclosed in this application is as follows.
Total mouse splenocytes are purified from spleens of 8-16 week old Balb/c mice by red blood cell lysis (BD Pharmingen #555899). Testing compounds are diluted to 0.5% DMSO and incubated with 1.25×106 splenocytes in a final volume of 200 μl in flat clear bottom plates (Falcon 353072) for 60 min at 37° C. Cells are then stimulated with the addition of 15 μg/ml IgM (Jackson ImmunoResearch 115-006-020), and incubated for 16 hr at 37° C., 5% CO2. Following the 16 hr incubation, cells are transferred to conical bottom clear 96-well plates and pelleted by centrifugation at 1200×g×5 min. Cells are preblocked by CD16/CD32 (BD Pharmingen #553142), followed by triple staining with CD19-FITC (BD Pharmingen #553785), CD69-PE (BD Pharmingen #553237), and 7AAD (BD Pharmingen #51-68981E). Cells are sorted on a BD FACSCalibur and gated on the CD19+/7AAD− population. The levels of CD69 surface expression on the gated population is thus determined. If CD69 expression is determined following treatment with different concentrations of test compound, CD69 expression level can be plotted versus test compound concentration.
Example 9 BMMC Degranulation
A generalized procedure for a standard assay for bone-marrow derived mouse mast cell (BMMC) degranulation that can be used to test compounds disclosed in this application is as follows.
Bone-marrow derived mast cells were cultured for >4 weeks with IL-3 (10 ng/ml) and SCF (10 ng/ml). The cells were determined to be >90% cKit+/FceRI+ by FACS analysis at the time of use. Cells (6×107 cells/50 ml) were serum-starved in a T150 tissue culture flask for 16 h in the absence of IL-3 and SCF containing IgE anti-DNP at 1 ug/ml. Overnight sensitized cells are washed twice in Tyrodes buffer and resuspended to 5×106 cells/ml. 5×105 cells (100 ul) are plated in a 96 well microtiter plate (Falcon 353072) and test compounds are serially diluted to a final concentration 0.25% DMSO in the plate for 1 hr at 37° C., 5% CO2. Wells are treated with a DNP-BSA antigen challenge (50 ng/ml) and incubated for and additional 30 min at 37° C. Supernatants are assayed for hexosamimidase release versus control wells. Cell pellets are simultaneously lysed and assessed for total hexosamimidase release to calculate specific release. Dose-response curves are generated using 4-parameter logistical fit and IC50s calculated.
Example 10 Passive Cutaneous Anaphylaxis (PCA)
The following is a procedure for a standard PCA model used for measuring in vivo IgE anti-DNP Ab sensitization and DNP-BSA antigen for triggering mast cell degranulation and release of immune regulators that cause acute vessel permeability monitored by Evan's blue dye into the inflamed area in the mouse ear.
Reagents: IgE anti-DNP: is supplied as 1.2 mg/ml in a phosphate buffered solution with BSA for additional protein and azide for sterility. This is diluted 1:100 in sterile PBS as a 12 ug/ml working stock that can be further diluted in PBS to the appropriate concentration for injection. A further 1:5 dilution give a final 1:500 solution at 2.4 ng/ul. (10 ul/ear=24 ng). Sterile PBS alone will be used as a negative control. -DNP-BSA: will be made up at 4 mg/ml in sterile ddH2O and stored at 40° C. solution. It is further diluted 1:1 with sterile saline prior to use. This solution or a further dilution in saline is diluted 1:1 with 2% Evan's Blue in sterile saline that has been filtered though 0.02 um filter and refiltered prior to injection. For these experiments a final solution of 0.5 mg/ml of DNP-BSA in 1% Evans blue can be used. Tail vein injections will be held constant at 200 ul=100 ug in 1% Evan's Blue. -Evan's blue dye: A 2% stock in saline will be sterile filtered and diluted 1:1 with DNP-BSA saline solution for final concentration of 1% for injection.
General PCA Protocol Using Intradermal Ear Sensitization
1) On d0, animals, anesthetized with isofluorine, are passively sensitized by intradermal injections of IgE anti-DNP using a 29-gauge insulin syringe. By convention, the right ear receives 10 ul intradermal injection of IgE anti-DNP while the left ear receives PBS.
2) 20 hr post sensitization, antigen challenge is administered by tail i.v. injection of DNP-BSA in 200 ul of 1% Evans blue dye solution in saline. Tails are immersed in warm water prior to iv injection to improve success. 3) 30 minutes to 2 hr prior to this antigen challenge, drug is delivered sc or po in 10% EtOH/20% cremaphor/70% saline. 4) Animals are sacrifice by CO2 inhalation 30-60 min post antigen challenge and ears are removed for extraction of Evan's blue dye in 500 ul of formamide overnight at 65° C. 5) Blood is obtained by cardiac puncture just prior to final cervical dislocation and processed for plasma to provide PK analysis. 6) Evan's blue dye is quantified by reading absorbency of 200 ul of extracted solution in microtiter plates at 620 nm.
Study Design of Experiment
Each animal has one IgE anti-DNP sensitized ear (right ear by convention) and one PBS control ear (left ear by convention). Groups 1-8: represent the vehicle and compound testing arms; Group 9: represents the non-antigen negative control; Group 10: represents the non-sensitized challenged negative control; Group 11: represents the non-antigen challenged, non-sensitized negative control group (Groups 9-11 represent negative controls for background levels only and require only minimal number of animals per group.)
The compounds disclosed in the examples above were tested in the Syk biochemical assay described herein (Example 4) and certain of those compounds exhibited an IC50 value less than or equal to 1 micromolar. Certain of those compounds exhibited an IC50 value less than or equal to 100 nM. Certain of those compounds exhibited an IC50 value less than or equal to 10 nM.
Some of the compounds disclosed in synthetic Example 3 were tested in the B-cell proliferation assay (as described in Example 6) and exhibited an IC50 value less than or equal to 10 micromolar. Certain of those compounds exhibited an IC50 value less than or equal to 1 micromolar.
Certain of those compounds did not inhibit T-cell proliferation and had IC50 values greater than or equal to 5 micromolar when assayed under conditions described herein (as described in Example 7).
Certain compounds described herein exhibited IC50 values for inhibition of T-cell proliferation that were at least 3-fold, and in some instances 5-fold, greater than the IC50 values of those compounds for inhibition of B-cell proliferation.
Some of the compounds described herein were tested in an assay for inhibition of B cell activity (under the conditions described in example 8), and exhibited an IC50 value less than or equal to 10 micromolar. Certain of those compounds exhibited an IC50 value less than or equal to 1 micromolar.
Some of the compounds disclosed in described herein exhibited both biochemical and cell-based activity. For example, some of the compounds described herein exhibited an IC50 value less than or equal to 10 micromolar in the Syk biochemical assay described herein (Example 4) and an IC50 value less than or equal to 10 micromolar in at least one of the cell-based assays (other than the T-cell assay) described herein (Examples 5, 6, 8 or 9). Certain of those compounds exhibited an IC50 value less than or equal to 1 micromolar in the Syk biochemical assay described herein (Example 4) and an IC50 value less than or equal to 10 micromolar in at least one of the cell-based assays (other than the T-cell assay) described herein (Examples 5, 6, 8 or 9). Certain of those compounds exhibited an IC50 value less than or equal to 0.1 micromolar and an IC50 value less than or equal to 10 micromolar in at least one of the cell-based assays (other than the T-cell assay) described herein (Examples 5, 6, 8 or 9).
While some embodiments have been shown and described, various modifications and substitutions may be made thereto without departing from the spirit and scope of the invention. For example, for claim construction purposes, it is not intended that the claims set forth hereinafter be construed in any way narrower than the literal language thereof, and it is thus not intended that exemplary embodiments from the specification be read into the claims. Accordingly, it is to be understood that the present invention has been described by way of illustration and not limitations on the scope of the claims.

Claims (7)

What is claimed:
1. A compound chosen from
Figure US08697699-20140415-C00161
or a pharmaceutically acceptable salt thereof.
2. A pharmaceutical composition comprising a compound of claim 1, together with at least one pharmaceutically acceptable vehicle chosen from carriers, adjuvants and excipients.
3. A method for inhibiting ATP hydrolysis, the method comprising contacting cells expressing Syk with a compound of claim 1 in an amount sufficient to detectably decrease the level of ATP hydrolysis in vitro.
4. The method of claim 3 wherein the cells are present in a mammal.
5. The method of claim 4 wherein the mammal is a human.
6. A method for determining the presence of Syk in a sample, comprising contacting the sample with a compound of claim 1 under conditions that permit detection of Syk activity, detecting a level of Syk activity in the sample, and therefrom determining the presence or absence of Syk in the sample.
7. A method for inhibiting B-cell activity comprising contacting cells expressing Syk with a compound of claim 1, in an amount sufficient to detectably decrease B-cell activity in vitro.
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